Antibody fragment-polymer conjugates and uses of same

ABSTRACT

Described are conjugates formed by an antibody fragment covalently attached to a non-proteinaceous polymer, wherein the apparent size of the conjugate is at least about 500 kD. The conjugates exhibit substantially improved half-life, mean residence time, and/or clearance rate in circulation as compared to the underivatized parental antibody fragment. Also described are conjugates directed against human vascular endothelial growth factor (VEGF), human p185 receptor-like tyrosine kinase (HER2), human CD20, human CD18, human CD11a, human IgE, human apoptosis receptor-2 (Apo-2), human tumor necrosis factor-α (TNF-α), human tissue factor (TF), human α4β7 integrin, human GPIIb-IIIa integrin, human epidermal growth factor receptor (EGFR), human CD3, and human interleukin-2 receptor α-chain (TAC) for diagnostic and therapeutic applications.

This application is a continuation of U.S. patent application Ser. No.09/489,394, filed Jan. 21, 2000 now U.S. Pat. No. 7,122,636, thecontents of which are incorporated herein by reference, which is anon-provisional application filed under 37 C.F.R. §1.53(b)(1), claimingpriority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser.No. 60/116,787, filed Jan. 21, 1999, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

This application relates to the field of antibody fragments derivatizedwith polymers, and in particular to the use of such derivatization toincrease the circulation half-lives of antibody fragment-polymerconjugates. This application also relates to the use of such antibodyfragment-polymer conjugates in the treatment of diseases.

BACKGROUND

Modification of proteins with polyethylene glycol (“PEGylation”) has thepotential to increase residence time and reduce immunogenicity in vivo.For example, Knauf et al., J. Biol. Chem., 263: 15064–15070 (1988)reported a study of the pharmacodynamic behavior in rats of variouspolyoxylated glycerol and polyethylene glycol modified species ofinterleukin-2. Despite the known advantage of PEGylation, PEGylatedproteins have not been widely exploited for clinical applications. Inthe case of antibody fragments, PEGylation has not been shown to extendserum half-life to useful levels. Delgado et al., Br. J. Cancer, 73:175–182 (1996), Kitamura et al., Cancer Res., 51: 4310–4315 (1991),Kitamura et al., Biochem. Biophys. Res. Comm., 171: 1387–1394 (1990),and Pedley et al., Br. J. Cancer, 70: 1126–1130 (1994) reported studiescharacterizing blood clearance and tissue uptake of certain anti-tumorantigen antibodies or antibody fragments derivatized with low molecularweight (5 kD) PEG. Zapata et al., FASEB J. 9: A1479 (1995) reported thatlow molecular weight (5 or 10 kD) PEG attached to a sulfhydryl group inthe hinge region of a Fab′ fragment reduced clearance compared to theparental Fab′ molecule.

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors,intraocular neovascular syndromes such as proliferative retinopathies orage-related macular degeneration (AMD), rheumatoid arthritis, andpsoriasis (Folkman et al. J. Biol. Chem. 267:10931–10934 (1992);Klagsbrun et al. Annu. Rev. Physiol. 53:217–239 (1991); and Garner A,Vascular diseases. In: Pathobiology of ocular disease. A dynamicapproach. Garner A, Klintworth G K, Eds. 2nd Edition Marcel Dekker, NY,pp 1625–1710 (1994)). In the case of solid tumors, theneovascularization allows the tumor cells to acquire a growth advantageand proliferative autonomy compared to the normal cells. Accordingly, acorrelation has been observed between density of microvessels in tumorsections and patient survival in breast cancer as well as in severalother tumors (Weidner et al. N Engl J Med 324:1–6 (1991); Horak et al.Lancet 340:1120–1124 (1992); and Macchiarini et al. Lancet 340:145–146(1992)).

Work done over the last several years has established the key role ofvascular endothelial growth factor (VEGF) in the regulation of normaland abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18:4–25 (1997)).The finding that the loss of even a single VEGF allele results inembryonic lethality points to an irreplaceable role played by thisfactor in the development and differentiation of the vascular system(Ferrara et al.). Furthermore, VEGF has been shown to be a key mediatorof neovascularization associated with tumors and intraocular disorders(Ferrara et al.). The VEGF mRNA is overexpressed by the majority ofhuman tumors examined (Berkman et al. J Clin Invest 91:153–159 (1993);Brown et al. Human Pathol. 26:86–91 (1995); Brown et al. Cancer Res.53:4727–4735 (1993); Mattem et al. Brit. J. Cancer. 73:931–934 (1996);and Dvorak et al. Am J. Pathol. 146:1029–1039 (1995)). Also, theconcentration of VEGF in eye fluids are highly correlated to thepresence of active proliferation of blood vessels in patients withdiabetic and other ischemia-related retinopathies (Aiello et al. N.Engl. J. Med. 331:1480–1487 (1994)). Furthermore, recent studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855–868 (1996)). Anti-VEGF neutralizing antibodies suppress thegrowth of a variety of human tumor cell lines in nude mice (Kim et al.Nature 362:841–844 (1993); Warren et al. J. Clin. Invest. 95:1789–1797(1995); Borgström et al. Cancer Res. 56:4032–4039 (1996); and Melnyk etal. Cancer Res. 56:921–924 (1996)) and also inhibit intraocularangiogenesis in models of ischemic retinal disorders (Adamis et al.Arch. Ophihalmol. 114:66–71 (1996)). Therefore, anti-VEGF monoclonalantibodies or other inhibitors of VEGF action are promising candidatesfor the treatment of solid tumors and various intraocular neovasculardisorders.

Proto-oncogenes that encode growth factors and growth factor receptorshave been identified to play important roles in the pathogenesis ofvarious human malignancies, including breast cancer. It has been foundthat the human erbB2 gene (also known as HER2, or c-erbB-2), whichencodes a 185-kd transmembrane glycoprotein receptor (p185^(HER2))related to the epidermal growth factor receptor (EGFR), is overexpressedin about 25% to 30% of human breast cancer (Slamon et al., Science235:177–182 [1987]; Slamon et al., Science 244:707–712 [1989]).

Several lines of evidence support a direct role for ErbB2 in thepathogenesis and clinical aggressiveness of ErbB2-overexpressing tumors.The introduction of ErbB2 into non-neoplastic cells has been shown tocause their malignant transformation (Hudziak et al., Proc. Natl. Acad.Sci. USA 84:7159–7163 [1987]; DiFiore et al., Science 237:78–182[1987]). Transgenic mice that express HER2 were found to develop mammarytumors (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578–10582 [1992]).

Antibodies directed against human erbB2 protein products and proteinsencoded by the rat equivalent of the erbB2 gene (neu) have beendescribed. Drebin et al., Cell 41:695–706 (1985) refer to an IgG2amonoclonal antibody which is directed against the rat neu gene product.This antibody called 7.16.4 causes down-modulation of cell surface p185expression on B104-1-1 cells (NIH-3T3 cells transfected with the neuprotooncogene) and inhibits colony formation of these cells. In Drebinet al. PNAS (USA) 83:9129–9133 (1986), the 7.16.4 antibody was shown toinhibit the tumorigenic growth of neu-transformed NIH-3T3 cells as wellas rat neuroblastoma cells (from which the neu oncogene was initiallyisolated) implanted into nude mice. Drebin et al. in Oncogene 2:387–394(1988) discuss the production of a panel of antibodies against the ratneu gene product. All of the antibodies were found to exert a cytostaticeffect on the growth of neu-transformed cells suspended in soft agar.Antibodies of the IgM, IgG2a and IgG2b isotypes were able to mediatesignificant in vitro lysis of neu-transformed cells in the presence ofcomplement, whereas none of the antibodies were able to mediate highlevels of antibody-dependent cellular cytotoxicity (ADCC) of theneu-transformed cells. Drebin et al. Oncogene 2:273–277 (1988) reportthat mixtures of antibodies reactive with two distinct regions on thep185 molecule result in synergistic anti-tumor effects onneu-transformed NIH-3T3 cells implanted into nude mice. Biologicaleffects of anti-neu antibodies are reviewed in Myers et al., Meth.Enzym. 198:277–290 (1991). See also WO94/22478 published Oct. 13, 1994.

Hudziak et al., Mol. Cell. Biol. 9(3):1165–1172 (1989) describe thegeneration of a panel of anti-ErbB2 antibodies which were characterizedusing the human breast tumor cell line SKBR3. Relative cellproliferation of the SKBR3 cells following exposure to the antibodieswas determined by crystal violet staining of the monolayers after 72hours. Using this assay, maximum inhibition was obtained with theantibody called 4D5 which inhibited cellular proliferation by 56%. Otherantibodies in the panel, including 7C2 and 7F3, reduced cellularproliferation to a lesser extent in this assay. Hudziak et al. concludethat the effect of the 4D5 antibody on SKBR3 cells was cytostatic ratherthan cytotoxic, since SKBR3 cells resumed growth at a nearly normal ratefollowing removal of the antibody from the medium. The antibody 4D5 wasfurther found to sensitize p185^(erbB2)-overexpressing breast tumor celllines to the cytotoxic effects of TNF-. See also WO89/06692 publishedJul. 27, 1989. The anti-ErbB2 antibodies discussed in Hudziak et al. arefurther characterized in Fendly et al. Cancer Research 50:1550–1558(1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. GrowthRegulation 1:72–82 (1991); Shepard et al. J. Clin. Immunol.11(3):117–127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979–986(1991); Lewis et al. Cancer Immunol. Immunother. 37:255–263 (1993);Pietras et al. Oncogene 9:1829–1838 (1994); Vitetta et al. CancerResearch 54:5301–5309 (1994); Sliwkowski et al. J. Biol. Chem.269(20):14661–14665 (1994); Scott et al. J. Biol. Chem. 266:14300–5(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202–7206 (1994).

Tagliabue et al. Int. J. Cancer 47:933–937 (1991) describe twoantibodies which were selected for their reactivity on the lungadenocarcinoma cell line (Calu-3) which overexpresses ErbB2. One of theantibodies, called MGR3, was found to internalize, inducephosphorylation of ErbB2, and inhibit tumor cell growth in vitro.

McKenzie et al. Oncogene 4:543–548 (1989) generated a panel ofanti-ErbB2 antibodies with varying epitope specificities, including theantibody designated TA1. This TA1 antibody was found to induceaccelerated endocytosis of ErbB2 (see Maier et al. Cancer Res.51:5361–5369 (1991)). Bacus et al. Molecular Carcinogenesis 3:350–362(1990) reported that the TA1 antibody induced maturation of the breastcancer cell lines AU-565 (which overexpresses the erbB2 gene) and MCF-7(which does not). Inhibition of growth and acquisition of a maturephenotype in these cells was found to be associated with reduced levelsof ErbB2 receptor at the cell surface and transient increased levels inthe cytoplasm.

Stancovski et al. PNAS (USA) 88:8691–8695 (1991) generated a panel ofanti-ErbB2 antibodies, injected them i.p. into nude mice and evaluatedtheir effect on tumor growth of murine fibroblasts transformed byoverexpression of the erbB2 gene. Various levels of tumor inhibitionwere detected for four of the antibodies, but one of the antibodies(N28) consistently stimulated tumor growth. Monoclonal antibody N28induced significant phosphorylation of the ErbB2 receptor, whereas theother four antibodies generally displayed low or nophosphorylation-inducing activity. The effect of the anti-ErbB2antibodies on proliferation of SKBR3 cells was also assessed. In thisSKBR3 cell proliferation assay, two of the antibodies (N12 and N29)caused a reduction in cell proliferation relative to control. Theability of the various antibodies to induce cell lysis in vitro viacomplement-dependent cytotoxicity (CDC) and antibody-mediatedcell-dependent cytotoxicity (ADCC) was assessed, with the authors ofthis paper concluding that the inhibitory function of the antibodies wasnot attributed significantly to CDC or ADCC.

Bacus et al. Cancer Research 52:2580–2589 (1992) further characterizedthe antibodies described in Bacus et al. (1990) and Stancovski et al. ofthe preceding paragraphs. Extending the i.p. studies of Stancovski etal., the effect of the antibodies after i.v. injection into nude miceharboring mouse fibroblasts overexpressing human ErbB2 was assessed. Asobserved in their earlier work, N28 accelerated tumor growth whereas N12and N29 significantly inhibited growth of the ErbB2-expressing cells.Partial tumor inhibition was also observed with the N24 antibody. Bacuset al. also tested the ability of the antibodies to promote a maturephenotype in the human breast cancer cell lines AU-565 and MDA-MB453(which overexpress ErbB2) as well as MCF-7 (containing low levels of thereceptor). Bacus et al. saw a correlation between tumor inhibition invivo and cellular differentiation; the tumor-stimulatory antibody N28had no effect on differentiation, and the tumor inhibitory action of theN12, N29 and N24 antibodies correlated with the extent ofdifferentiation they induced.

Xu et al. Int. J. Cancer 53:401–408 (1993) evaluated a panel ofanti-ErbB2 antibodies for their epitope binding specificities, as wellas their ability to inhibit anchorage-independent andanchorage-dependent growth of SKBR3 cells (by individual antibodies andin combinations), modulate cell-surface ErbB2, and inhibit ligandstimulated anchorage-independent growth. See also WO94/00136 publishedJan. 6, 1994 and Kasprzyk et al. Cancer Research 52:2771–2776 (1992)concerning anti-ErbB2 antibody combinations. Other anti-ErbB2 antibodiesare discussed in Hancock et al. Cancer Res. 51:4575–4580 (1991); Shawveret al. Cancer Res. 54:1367–1373 (1994); Arteaga et al. Cancer Res.54:3758–3765 (1994); and Harwerth et al. J. Biol. Chem. 267:15160–15167(1992).

A recombinant humanized anti-ErbB2 monoclonal antibody (a humanizedversion of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAbHER2 or HERCEPTIN®) has been clinically active in patients withErbB2-overexpressing metastatic breast cancers that had receivedextensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol.14:737–744 [1996]).

ErbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159:19–27 [1995]; and Hynes and Stern, BiochimBiophys Acta 1198:165–184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 11(3 Suppl 1):43–48 [1997]).However, despite the association of ErbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients (Ibid). rhuMab HER2 was shown to enhance theactivity of paclitaxel (TAXOL®) and doxorubicin against breast cancerxenografts in nude mice injected with BT-474 human breast adenocarcinomacells, which express high levels of HER2 (Baselga et al., Breast Cancer,Proceedings of ASCO, Vol. 13, Abstract 53 [1994]).

Lymphocyte adherence to endothelium is a key event in the process ofinflammation. There are at least three known pathways of lymphocyteadherence to endothelium, depending on the activation state of the Tcell and the endothelial cell. T cell immune recognition requires thecontribution of the T cell receptor as well as adhesion receptors, whichpromote attachment of T cells to antigen-presenting cells and transduceregulatory signals for T cell activation. The lymphocyte functionassociated (LFA) antigen-1 (LFA-1, CD11a, α-chain/CD18, β-chain) hasbeen identified as the major integrin receptor on lymphocytes involvedin these cell adherence interactions leading to several pathologicalstates. ICAM-1, the endothelial cell immunoglobulin-like adhesionmolecule, is a known ligand for LFA-1 and is implicated directly ingraft rejection, psoriasis, and arthritis.

LFA-1 is required for a range of leukocyte functions, includinglymphokine production of helper T cells in response toantigen-presenting cells, killer T cell-mediated target cell lysis, andimmunoglobulin production through T cell-B cell interactions. Activationof antigen receptors on T cells and B cells allows LFA-1 to bind itsligand with higher affinity.

Monoclonal antibodies (MAbs) directed against LFA-1 led to the initialidentification and investigation of the function of LFA-1. Davignon etal., J. Immunol., 127: 590 (1981). LFA-1 is present only on leukocytes[Krenskey et al., J. Immunol., 131: 611 (1983)], and ICAM-1 isdistributed on activated leukocytes, dermal fibroblasts, andendothelium. Dustin et al., J. Immunol., 137: 245 (1986).

Previous studies have investigated the effects of anti-CD11a MAbs onmany T-cell-dependent immune functions in vitro and a limited number ofimmune responses in vivo. In vitro, anti-CD11a MAbs inhibit T-cellactivation [Kuypers et al., Res. Immunol., 140: 461 (1989)],T-cell-dependent B-cell proliferation and differentiation [Davignon etal., supra; Fischer et al., J. Immunol., 136: 3198 (1986)], target celllysis by cytotoxic T lymphocytes [Krensky et al., supra], formation ofimmune conjugates (Sanders et al., J. Immunol., 137: 2395 (1986);Mentzer et al., J. Immunol., 135: 9 (1985)), and the adhesion of T-cellsto vascular endothelium. Lo et al., J. Immunol., 143: 3325 (1989). Also,the antibody 5C6 directed against CD11b/CD18 was found to preventintra-islet infiltration by both macrophages and T cells and to inhibitdevelopment of insulin-dependent diabetes mellitis in mice. Hutchings etal., Nature, 348: 639 (1990).

IgE is a member of the immunoglobulin family that mediates allergicresponses such as asthma, food allergies, and other type 1hypersensitivity reactions. IgE is secreted by and expressed on thesurface of B-cells or B-lymphocytes. IgE binds to B-cells (as well asmonocytes, eosinophils and platelets) through its Fc region to a lowaffinity IgE receptor (Fc_(∈)RII). Upon exposure of a mammal to anallergen, B-cells bearing a membrane-bound IgE antibody specific for theantigen are activated to form IgE-secreting plasma cells. Theallergen-specific, soluble IgE secreted by plasma cells circulatesthrough the bloodstream and binds to the surface of mast cells intissues and basophils in the blood, through the high affinity IgEreceptor (Fc_(∈)RI). The mast cells and basophils thereby becomesensitized for the allergen. Subsequent exposure to the allergen resultsin cross linking of allergen-specific IgE bound to basophilic and mastcellular Fc_(∈)RI, which induces a release of histamine, leukotrienesand platelet activating factors, eosinophil and neutrophil chemotacticfactors and the cytokines IL-3, IL-4, IL-5 and GM-CSF, which areresponsible for clinical hypersensitivity and anaphylaxis.

The pathological condition hypersensitivity is characterized by anexcessive immune response to (an) allergen(s) resulting in gross tissuechanges if the allergen is present in relatively large amounts or if thehumoral and cellular immune state is at a heightened level.

Physiological changes in anaphylactic hypersensitivity can includeintense constriction of the bronchioles and bronchi of the lungs,contraction of smooth muscle and dilation of capillaries. Predispositionto this condition appears to result from an interaction between geneticand environmental factors. Common environmental allergens which induceanaphylactic hypersensitivity are found in pollen, foods, house dustmites, animal danders, fungal spores and insect venoms. Atopic allergyis associated with anaphylactic hypersensitivity and includes disorderssuch as asthma, allergic rhinitis and conjunctivitis (hay fever),eczema, urticaria and food allergies. Anaphylactic shock, a dangerouslife-threatening condition that can occur in the progression ofanaphylaxis, is usually provoked by insect stings or parenteralmedication.

Recently, a treatment strategy has been pursued for Type Ihypersensitivity or anaphylactic hypersensitivity which blocks IgE frombinding to the high-affinity receptor (Fc_(∈)RI) found on basophils andmast cells, and thereby prevents the release of histamine and otheranaphylactic factors resulting in the pathological condition.

Interleukin-8 (IL-8) is neutrophil chemotactic peptide secreted by avariety of cells in response to inflammatory mediators (for a review seeHebert et al. Cancer Investigation 11(6):743 (1993)). IL-8 can play animportant role in the pathogenesis of inflammatory disorders, such asadult respiratory distress syndrome (ARDS), septic shock, and multipleorgan failure. Immune therapy for such inflammatory disorders caninclude treatment of an affected patient with anti-IL-8 antibodies.

Sticherling et al. (J. Immunol. 143:1628 (1989)) disclose the productionand characterization of four monoclonal antibodies against IL-8. WO92/04372, published Mar. 19, 1992, discloses polyclonal antibodies whichreact with the receptor-interacting site of IL-8 and peptide analogs ofIL-8, along with the use of such antibodies to prevent an inflammatoryresponse in patients. St. John et al. (Chest 103:932 (1993)) reviewimmune therapy for ARDS, septic shock, and multiple organ failure,including the potential therapeutic use of anti-IL-8 antibodies. Sekidoet al. (Nature 365:654 (1993)) disclose the prevention of lungreperfusion injury in rabbits by a monoclonal antibody against IL-8.Mulligan et al. (J. Immunol. 150:5585 (1993)), disclose protectiveeffects of a murine monoclonal antibody to human IL-8 in inflammatorylung injury in rats.

WO 95/23865 (International Application No. PCT/US95/02589 published Sep.8, 1995) demonstrates that anti-IL-8 monoclonal antibodies can be usedtherapeutically in the treatment of other inflammatory disorders, suchas bacterial pneumonias and inflammatory bowel disease.

Anti-IL-8 antibodies are additionally useful as reagents for assayingIL-8. For example, Sticherling et al. (Arch. Dermatol. Res. 284:82(1992)), disclose the use of anti-IL-8 monoclonal antibodies as reagentsin immunohistochemical studies. Ko et al. (J. Immunol. Methods 149:227(1992)) disclose the use of anti-IL-8 monoclonal antibodies as reagentsin an enzyme-linked immunoabsorbent assay (ELISA) for IL-8.

SUMMARY OF THE INVENTION

One aspect of the invention is a conjugate consisting essentially of oneor more antibody fragments covalently attached to one or morenonproteinaceous polymer molecules, wherein the apparent size of theconjugate is at least about 500 kD.

Another aspect of the invention is a conjugate formed by one or moreantibody fragments covalently attached to one or more nonproteinaceouspolymer molecules, wherein the apparent size of the conjugate is atleast about 500 kD, and wherein the covalent structure of the conjugateis free of any matter other than the antibody fragment andnonproteinaceous polymer molecules.

Yet another aspect of the invention is a conjugate formed by the one ormore antibody fragments covalently attached to one or morenonproteinaceous polymer molecules, wherein the covalent structure ofthe conjugate further incorporates one or more nonproteinaceous labels,wherein the covalent structure of the conjugate is free of any matterother than the antibody fragment, nonproteinaceous polymer andnonproteinaceous label molecules, and wherein the apparent size of theconjugate is at least about 500 kD.

Still another aspect of the invention is a conjugate consistingessentially of one or more antibody fragments covalently attached to oneor more nonproteinaceous polymer molecules, wherein the apparent size ofthe conjugate is at least about 500 kD, and wherein at least oneantibody fragment comprises an antigen binding site that binds to apolypeptide selected from the group consisting of human vascularendothelial growth factor (VEGF), human p185 receptor-like tyrosinekinase (HER2), human CD20, human CD18, human CD11a, human IgE, humanapoptosis receptor-2 (Apo-2), human tumor necrosis factor-α (TNF-α),human tissue factor, human α₄β₇ integrin, human GPIIb-IIIa integrin,human epidermal growth factor receptor (EGFR), human CD3, and humaninterleukin-2 receptor α-chain (TAC).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the blocking of IL-8 mediated elastaserelease from neutrophils by anti-IL-8 monoclonal antibody 5.12.14.

FIG. 2 is a graph depicting the inhibition of ¹²⁵I-IL-8 binding toneutrophils by unlabeled IL-8.

FIG. 3 demonstrates that a isotype matched negative control Fab (denotedas “4D5 Fab”) does not inhibit the binding of ¹²⁵I-IL-8 to humanneutrophils.

FIG. 4 is a graph depicting the inhibition of binding of ¹²⁵I-IL-8 tohuman neutrophils by chimeric 5.12.14 Fab with an average IC₅₀ of 1.6nM.

FIG. 5 is a graph depicting the inhibition of binding of ¹²⁵I-IL-8 tohuman neutrophils by chimeric 6G.4.25 Fab with an average IC₅₀ of 7.5nM.

FIG. 6 demonstrates the inhibition of human IL-8 mediated neutrophilchemotaxis by chimeric 6G4.2.5 Fab and chimeric 5.12.14 Fab.

FIG. 7 demonstrates the relative abilities of chimeric 6G4.2.5 Fab andchimeric 5.12.14 Fab to inhibit rabbit IL-8 mediated neutrophilchemotaxis.

FIG. 8 depicts the stimulation of elastase release from humanneutrophils by various concentrations of human and rabbit IL-8. Therelative extent of elastase release was quantitated by measurement ofabsorbance at 405 nm. The data represent mean±SEM of triplicate samples.

FIG. 9 is a graph depicting the ability of chimeric 6G4.2.5 Fab andchimeric 5.12.14 Fab to inhibit elastase release from human neutrophilsstimulated by human IL-8. The results were normalized to reflect thepercentage of elastase release elicited by 100 nM IL-8 alone. The datarepresent the mean±SEM of three separate experiments performed ondifferent days with different blood donors. IC₅₀ values were calculatedby four parameter fit.

FIG. 10 is a graph depicting the relative abilities of chimeric 6G4.2.5Fab and chimeric 5.12.14 Fab to inhibit elastase release from humanneutrophils stimulated by rabbit IL-8. The results were normalized toreflect the percentage of elastase release elicited by 100 nM IL-8alone. The data represent the mean±SEM of three separate experimentsperformed on different days with different blood donors. IC₅₀ valueswere calculated by four parameter fit.

FIGS. 11A–11J are a set of graphs depicting the following parameters ina rabbit ulcerative colitis model: FIG. 11A depicts myeloperoxidaselevels in tissue; FIG. 11B depicts IL-8 levels in tissue; FIG. 11Cdepicts colon weight; FIG. 11D depicts gross inflammation; FIG. 11Edepicts edema; FIG. 11F depicts extent of necrosis; FIG. 11G depictsseverity of necrosis; FIG. 11H depicts neutrophil margination; FIG. 11Idepicts neutrophil infiltration; and FIG. 11J depicts mononuclearinfiltration.

FIG. 12 is a graph depicting the effect of anti-IL-8 monoclonal antibodytreatment on the number of neutrophils in bronchoalveolar lavage (BAL)fluid in animals infected with Streptococcus pneumoniae, Escherichiacoli, or Pseudomonas aeruginosa. Treatment with 6G4.2.5 significantlyreduced the number of neutrophils present in the BAL fluid compared toanimals treated with isotype control mouse IgG (FIG. 12).

FIG. 13 depicts the DNA sequences (SEQ ID NOS: 1–6) of three primersdesigned for each of the light and heavy chains. Multiple primers weredesigned in order to increase the chances of primer hybridization andefficiency of first strand cDNA synthesis for cloning the variable lightand heavy regions of monoclonal antibody 5.12.14.

FIG. 14 depicts the DNA sequences (SEQ ID NOS: 7–10) of one forwardprimer and one reverse primer for the 5.12.14 light chain variableregion amplification.

FIG. 15 depicts the DNA sequences (SEQ ID NOS: 11–15) of one forwardprimer and one reverse primer for the 5.12.14 heavy chain variableregion amplification.

FIG. 16 depicts the DNA sequence (SEQ ID NO: 16) and the amino acidsequence (SEQ ID NO: 17) of the 5.12.14 light chain variable region andpartial murine constant light region. CDRs are indicated by either X-raycrystallography (underlined amino acids) or by Kabat sequence comparison(amino acids denoted with asterisk). Important restriction sites areindicated in italics. The signal peptide of STII is amino acids −23 to−1. The murine variable light region is amino acids 1 to 109. Thepartial murine constant light region is amino acids 110 to 123 (initalics).

FIG. 17 depicts the DNA sequence (SEQ ID NO: 18) and the amino acidsequence (SEQ ID NO: 19) of the 5.12.14 heavy chain variable region andpartial murine constant heavy region. CDRs are indicated by either X-raycrystallography (underlined amino acids) or by Kabat sequence comparison(amino acids denoted with asterisk). Important restriction sites areindicated in italics. The signal peptide of STII is amino acids −23 to−1. The murine variable heavy region is amino acids 1 to 120. Thepartial murine constant heavy region is amino acids 121 to 130.

FIG. 18 depicts the DNA sequences (SEQ ID NOS: 20–23) of amplificationprimers used to convert murine light and heavy chain constant regionresidues to their human equivalents.

FIG. 19 depicts the DNA sequence (SEQ ID NO: 24) and the amino acidsequence (SEQ ID NO: 25) for the 5.12.14 light chain variable region andthe human IgG1 light chain constant region. CDRs are indicated by eitherX-ray crystallography (underlined amino acids) or by Kabat sequencecomparison (amino acids denoted with asterisk). The human constantregion is denoted in italics. The signal peptide of STII is amino acids−23 to −1. The murine variable light region is amino acids 1 to 109. Thehuman constant light region is amino acids 110 to 215.

FIGS. 20A–20B depict the DNA sequence (SEQ ID NO: 26) and the amino acidsequence (SEQ ID NO: 27) for the 5.12.14 heavy chain variable region andthe heavy chain constant region of human IgG1. CDRs are indicated byeither X-ray crystallography (underlined amino acids) or by Kabatsequence comparison (amino acids denoted with asterisk). The humanconstant region is denoted in italics. The signal peptide of STII isamino acids −23 to −1. The murine variable heavy region is amino acids 1to 120. The human constant heavy region is amino acids 121 to 229.

FIG. 21 depicts the DNA sequences (SEQ ID NOS: 1–6) of three primersdesigned for each of the light and heavy chains. Multiple primers weredesigned in order to increase the chances of primer hybridization andefficiency of first strand cDNA synthesis for cloning the variable lightand heavy regions of monoclonal antibody 6G4.2.5.

FIG. 22 depicts the DNA sequences (SEQ ID NOS: 28–31) of one forwardprimer and one reverse primer for the 6G4.2.5 light chain variableregion amplification.

FIG. 23 depicts the DNA sequences (SEQ ID NOS: 32, 33, 11, 15, 14, and13) of one forward primer and one reverse primer for the 6G4.2.5 heavychain variable region amplification.

FIG. 24 depicts the DNA sequence (SEQ ID NO: 34) and the amino acidsequence (SEQ ID NO: 35) of the 6G4.2.5 light chain variable region andpartial murine constant light region. CDRs are indicated by either X-raycrystallography (underlined amino acids) or by Kabat sequence comparison(amino acids denoted with asterisk). Useful cloning sites are initalics. The signal peptide of STII is amino acids −23 to −1. The murinevariable light region is amino acids 1 to 114. The partial murineconstant light region is amino acids 115 to 131.

FIG. 25 depicts the DNA sequence (SEQ ID NO: 36) and the amino acidsequence (SEQ ID NO: 37) of the 6G4.2.5 heavy chain variable region andpartial murine constant heavy region. CDRs are indicated by either X-raycrystallography (underlined amino acids) or by Kabat sequence comparison(amino acids denoted with asterisk). Useful cloning sites are initalics. The signal peptide of STII is amino acids −23 to −1. The murinevariable heavy region is amino acids 1 to 122. The partial murineconstant heavy region is amino acids 123 to 135.

FIG. 26 depicts the DNA sequences (SEQ ID NOS: 38–40) of primers toconvert the murine light chain and heavy chain constant regions to theirhuman equivalents.

FIGS. 27A–27B depict the DNA sequence (SEQ ID NO: 41) and the amino acidsequence (SEQ ID NO: 42) for the chimeric 6G4.2.5 light chain. CDRs areindicated by either X-ray crystallography (underlined amino acids) or byKabat sequence comparison (amino acids denoted with asterisk). The humanconstant region is denoted in italics. The signal peptide of STII isamino acids −23 to −1. The murine variable light region is amino acids 1to 114. The human constant light region is amino acids 115 to 220.

FIGS. 28A–28B depict the DNA sequence (SEQ ID NO: 43) and the amino acidsequence (SEQ ID NO: 44) for the chimeric 6G4.2.5 heavy chain. CDRs areindicated by either X-ray crystallography (underlined amino acids) or byKabat sequence comparison (amino acids denoted with asterisk). The humanconstant region is denoted in italics. The signal peptide of STII isamino acids −23 to −1. The murine variable heavy region is amino acids 1to 122. The human constant heavy region is amino acids 123 to 231.

FIG. 29 depicts an amino acid sequence alignment of murine 6G425 lightchain variable domain (SEQ ID NO: 45), humanized 6G425 F(ab)-1 lightchain variable domain (SEQ ID NO: 46), and human light chain κIconsensus framework (SEQ ID NO: 47) amino acid sequences, and an aminoacid sequence alignment of murine 6G425 heavy chain variable domain (SEQID NO: 48), humanized 6G425 F(ab)-1 heavy chain variable domain (SEQ IDNO: 49), and human IgG1 subgroup III heavy chain variable domain (SEQ IDNO: 50) amino acid sequences, used in the humanization of 6G425. Lightchain CDRs are labeled L1, L2, L3; heavy chain CDRs are labeled H1, H2,and H3. = and + indicate CDR sequences as defined by X-raycrystallographic contacts and sequence hypervariability, respectively. #indicates a difference between the aligned sequences. Residue numberingis according to Kabat et al. Lower case lettering denotes the insertionof an amino acid residue relative to the humIII consensus sequencenumbering.

FIGS. 30A, 30B and 30C are graphs depicting the ability of F(ab)-9(humanized 6G4V11 Fab) to inhibit human wild type IL-8, human monomericIL-8, and rhesus IL-8 mediated neutrophil chemotaxis, respectively. FIG.30A presents inhibition data for F(ab)-9 samples at concentrations of0.06 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM, for an isotypecontrol antibody (denoted “4D5”) sample at a concentration of 100 nM,and for a no antibody control sample, in the presence of 2 nM human wildtype IL-8. FIG. 30B presents inhibition data for F(ab)-9 samples atconcentrations of 6.25 nM, 12.5 nM, 25 nM, and 50 nM, for an isotypecontrol antibody (denoted “4D5”) sample at a concentration of 100 nM,and for a no antibody control sample, in the presence of 4 nM humanmonomeric IL-8 (denoted as “BD59” and as “monomeric IL-8”). FIG. 30Cpresents inhibition data for F(ab)-9 samples at concentrations of 1 nM,12.5 nM, 25 nM, and 50 nM, for an isotype control antibody (denoted“4D5”) sample at a concentration of 100 nM, and for a no antibodycontrol sample, in the presence of 2 nM rhesus IL-8. In addition, FIGS.30A–30C each presents data for a no IL-8 buffer control sample (denotedas “Buffer”) in the respective inhibition assay.

FIG. 31A depicts the amino acid sequences of the humanized anti-IL-86G4.2.5V11 light chain in an N-terminal fusion with the STII leaderpeptide (SEQ ID NO: 51), the humanized anti-IL-8 6G4.2.5V11 heavy chainin an N-terminal fusion with the STII leader peptide (SEQ ID NO: 52),and a peptide linker in a C-terminal fusion with M13 phage gene-III coatprotein (SEQ ID NO: 53).

FIG. 31B depicts the nucleic acid sequence (SEQ ID NO: 54) and thetranslated amino acid sequence (SEQ ID NO: 51) of the humanizedanti-IL-8 6G4.2.5V11 light chain in an N-terminal fusion with the STIIleader peptide.

FIG. 31C depicts the amino acid sequences of the humanized anti-IL-86G4.2.5V19 light chain in an N-terminal fusion with the STII leaderpeptide (SEQ ID NO: 51), and the humanized anti-IL-8 6G4.2.5V19 heavychain in an N-terminal fusion with the STII leader peptide (SEQ ID NO:55).

FIG. 32 is a three dimensional computer model of the humanized anti-IL-86G4.2.5V11 antibody. Heavy chain CDR loops and variable domain regionsappear in purple, and CDR-H3 side chain residues appear in yellow. Heavychain constant domain regions appear in red. Light chain CDR loops andvariable domain regions appear in off-white, and the Asn residue atamino acid position 35 (N35) in CDR L1 appears in green. Light chainconstant domain regions appear in amber.

FIG. 33 is a Scatchard plot depicting the inhibition of ¹²⁵I-IL-8binding to human neutrophils exhibited by intact murine 6G4.2.5 antibody(denoted 6G4 murine mAb), 6G4.2.5 murine-human chimera Fab (denoted 6G4chimera), humanized 6G4.2.5 Fab versions 1 and 11 (denoted V1 and V11),and variant 6G4.2.5V11N35A Fab (denoted V11N35A).

FIGS. 34A, 34B, 34C and 34D are graphs depicting the ability of6G4.2.5V11N35A Fab to inhibit human wild type IL-8, human monomericIL-8, rabbit IL-8, and rhesus IL-8 mediated neutrophil chemotaxis,respectively. FIG. 34A presents inhibition data for 6G4.2.5V11N35A Fabsamples at concentrations of 0.5, 1, 2, 4, 8, 16, and 33 nM, for anisotype control antibody (denoted “4D5”) sample at a concentration of 33nM, and for a no antibody control (denoted “HuIL-8”) sample, in thepresence of 2 nM human wild type IL-8. FIG. 34B presents inhibition datafor 6G4.2.5V11N35A Fab samples at concentrations of 0.5, 1, 2, 4, 8, 16,and 33 nM, for an intact 6G4.2.5 mAb sample at a concentration of 33 nM,for an isotype control antibody (denoted as “4D5”) sample at aconcentration of 33 nM, and for a no antibody control (denoted “BD59”)sample, in the presence of 2 nM human monomeric IL-8. FIG. 34C presentsinhibition data for 6G4.2.5V11N35A Fab samples at concentrations of 0.5,1, 2, 4, 8, 16, and 33 nM, for an intact 6G4.2.5 mAb sample at aconcentration of 33 nM, for an isotype control antibody (denoted “4D5”)sample at a concentration of 33 nM, and for a no antibody control(denoted “Rab IL-8”) sample, in the presence of 2 nM rabbit IL-8. FIG.34D presents inhibition data for 6G4.2.5V11N35A Fab samples atconcentrations of 0.5, 1, 2, 4, 8, 16, and 33 nM, for an intact 6G4.2.5mAb sample at a concentration of 33 nM, for an isotype control antibody(denoted as “4D5”) sample at a concentration of 33 nM, and for a noantibody control (denoted “Rhe IL-8”) sample, in the presence of 2 nMrhesus IL-8. In addition, FIGS. 34B–34D each presents data for humanwild type IL-8 control (denoted “HuIL-8”) samples at a concentration of2 nM in the respective assay, and FIGS. 34A–34D each presents data for ano IL-8 buffer control (denoted “Buffer”) sample in the respectiveassay.

FIG. 35 depicts the amino acid sequences of the humanized anti-IL-86G4.2.5V11N35A light chain in an N-terminal fusion with the STII leaderpeptide (SEQ ID NO: 56), the humanized anti-IL-8 6G4.2.5V11N35A heavychain in an N-terminal fusion with the STII leader peptide (SEQ ID NO:52), and the GCN4 leucine zipper peptide (SEQ ID NO: 57). The Alaresidue (substituted for the wild type Asn residue) at amino acidposition 35 in the 6G4.2.5V11N35A light chain appears in bold case. Aputative pepsin cleavage site in the GCN4 leucine zipper sequence isunderlined.

FIG. 36 depicts the DNA sequence (SEQ ID NO: 58) and the amino acidsequence (SEQ ID NO: 56) of the humanized anti-IL-8 6G4.2.5V11N35A lightchain in an N-terminal fusion with the STII leader peptide.Complementarity determining regions L1, L2, and L3 are underlined

FIGS. 37A–37B depict the DNA sequence (SEQ ID NO: 59) and the amino acidsequence (SEQ ID NO: 60) of the humanized anti-IL-8 6G4.2.5V11N35A heavychain in an N-terminal fusion with the STII leader peptide and in aC-terminal fusion with the GCN4 leucine zipper sequence. Complementaritydetermining regions H1, H2, and H3 are underlined.

FIG. 38 is a Scatchard plot depicting the inhibition of ¹²⁵I-IL-8binding to human neutrophils exhibited by 6G4.2.5V11N35A Fab (denotedFab), 6G4.2.5V11N35A F(ab′)₂ (denoted F(ab′)₂), and human wild type IL-8control (denoted IL-8).

FIG. 39 is a graph depicting a comparison of the wild type human IL-8mediated neutrophil chemotaxis inhibition activities of the6G4.2.5V11N35A F(ab′)₂ and 6G4.2.5V11N35A Fab. Inhibition data arepresented for 6G4.2.5V11N35A Fab samples (denoted “N35A Fab”) and6G4.2.5V11N35A F(ab′)₂ samples (denoted N35A F(ab′)₂) at concentrationsof 0.3, 1, 3, 10, 30, and 100 nM, for an isotype control antibody(denoted as “4D5”) sample at a concentration of 100 nM, and for a noantibody control sample, in the presence of 2 nM human wild type IL-8.In addition, inhibition data are presented for no IL-8 buffer controlsamples (denoted “Buffer”).

FIG. 40 is a graph depicting the ability of 6G4.2.5V11N35A F(ab′)₂ toinhibit human monomeric IL-8, rhesus IL-8, and rabbit IL-8 mediatedneutrophil chemotaxis. Human monomeric IL-8 mediated neutrophilchemotaxis data are presented for 6G4.2.5V11N35A F(ab′)₂ samples atconcentrations of 0.3, 1, 3, and 10 nM, for an isotype control antibody(denoted as “4D5”) sample at a concentration of 100 nM, and for a noantibody control sample (denoted as “BD59”), in the presence of humanmonomeric IL-8 (denoted as “BD59”) at a concentration of 0.5 nM. RhesusIL-8 mediated neutrophil chemotaxis data are presented for6G4.2.5V11N35A F(ab′)₂ samples at concentrations of 0.3, 1, 3, and 10nM, and for a no antibody control sample, in the presence of rhesus IL-8at a concentration of 2 nM. Rabbit IL-8 mediated neutrophil chemotaxisdata are presented for 6G4.2.5V11N35A F(ab′)₂ samples at concentrationsof 0.3, 1, 3, and 10 nM, and for a no antibody control sample, in thepresence of rabbit IL-8 at a concentration of 2 nM. In addition,inhibition data are presented for a no IL-8 buffer control sample(denoted as “Buffer”) and for a 2 nM human wild type IL-8 (denoted as“HuIL-8”).

FIGS. 41A–41V depict the nucleic acid sequence (SEQ ID NO: 61) of thep6G4V11N35A.F(ab′)₂ vector.

FIG. 42 depicts the nucleic acid sequences of the stop template primer(SEQ ID NO: 63) and the NNS randomization primer (SEQ ID NO: 64) usedfor random mutagenesis of amino acid position 35 in variable light chainCDR-L1 of humanized antibody 6G4V11.

FIG. 43A is a table of data describing the frequencies of differentphage display clones obtained from the randomization of amino acidposition 35 in variable light chain CDR-L1 of humanized antibody 6G4V11.

FIGS. 43B, 43C, 43D and 43E are graphs of displacement curves depictingthe inhibition of ¹²⁵I-IL-8 binding to neutrophils exhibited by the6G4V11N35A, 6G4V11N35D, 6G4V11N35E and 6G4V11N35G Fab's.

FIG. 44 contains a graph depicting the typical kinetics of an anti-IL-8antibody fragment (6G4V11N35A F(ab′)₂) binding to IL-8. FIG. 44 alsocontains a table of data providing the equilibrium constant for6G4V11N35A Fab binding to IL-8 (rate constants were not determined“ND”), and the equilibrium and rate constants for 6G4V11N35A F(ab′)₂ and6G4V11N35E Fab binding to IL-8.

FIG. 45 depicts the DNA sequence (SEQ ID NO: 65) and amino acid sequence(SEQ ID NO: 62) of the 6G4V11N35E light chain in an N-terminal fusionwith the STII leader peptide.

Complementarity determining regions L1, L2 and L3 are underlined.

FIG. 46 is a graph depicting the ability of 6G4V11N35E Fab to inhibithuman IL-8 (dark columns) and rabbit IL-8 (light columns) mediatedneutrophil chemotaxis. Data are presented for 6G4V11N35E Fab samples atconcentrations of 0.4, 1.2, 3.7, 11 and 33 nM, and for an isotypecontrol antibody (4D5) sample at a concentration of 100 nM, in thepresence of 2 nM human IL-8 or 2 nM rabbit IL-8. In addition, inhibitiondata are presented for a no IL-8 buffer control sample (denoted“Buffer”) and for human and rabbit IL-8 control samples (denoted“IL-8”).

FIG. 47 depicts the DNA sequence of the sense (SEQ ID NO: 66) andanti-sense (SEQ ID NO: 67) strands of a PvuII-XhoI synthetic nucleotideencoding amino acids Leu4 to Phe29 of the 6G4V11N35A heavy chain.

FIGS. 48A–48T depict the DNA sequence (SEQ ID NO: 68) of plasmidp6G4V11N35A.choSD9.

FIGS. 49A, 49B, 49C and 49D are graphs of displacement curves depictingthe inhibition of ¹²⁵I-IL-8 binding to neutrophils exhibited by IL-8control, intact murine 6G4.2.5 antibody, the full length IgG1 form ofvariant 6G4V11N35A, and the full length IgG1 form of variant 6G4V11N35E,respectively.

FIGS. 50A–50B are graphs depicting the ability of full length 6G4V11N35AIgG1 and 6G4V11N35E IgG1 to inhibit human IL-8 (FIG. 50A) and rabbitIL-8 (FIG. 50B) mediated neutrophil chemotaxis.

FIG. 51 contains a graph depicting the typical kinetics of a full lengthanti-IL-8 antibody (6G4V11N35A IgG1) binding to IL-8. FIG. 51 alsocontains a table of data providing the equilibrium and rate constantsfor full length murine 6G4.2.5 IgG2a, 6G4V11N35A IgG1 and 6G4V11N35EIgG1 binding to IL-8.

FIGS. 52A and 52B are graphs of displacement curves depicting theresults of an unlabeled IL-8/¹²⁵I-IL-8 competition radioimmunoassayperformed with full length 6G4V11N35A IgG1 and 6G4V11N35E IgG1,respectively.

FIG. 53 depicts the DNA sequence (SEQ ID NO: 69) and amino acid sequence(SEQ ID NO: 70) of the 6G4V11N35A Fab′ heavy chain (6G4V11N35A Fab heavychain modified to contain a cysteine residue in the hinge region).

FIGS. 54A–54C contain graphs of displacement curves depicting the IL-8binding and IC₅₀'s for PEG-maleimide modified 6G4V11N35A Fab′ molecules.

FIGS. 55A–55C are graphs depicting the ability of PEG-maleimide modified6G4V11N35A Fab′ molecules to inhibit human IL-8 and rabbit IL-8 mediatedneutrophil chemotaxis.

FIGS. 56A–56C are graphs depicting the ability of PEG-maleimide modified6G4V11N35A Fab′ molecules to inhibit IL-8 mediated release ofP-glucuronidase from neutrophils.

FIGS. 57A–57B contain graphs of displacement curves depicting theinhibition of ¹²⁵I-IL-8 binding to neutrophils exhibited byPEG-succinimide modified 6G4V11N35A Fab′₂ molecules.

FIGS. 58A–58B are graphs depicting the ability of PEG-succinimidemodified 6G4V11N35A F(ab′)₂ molecules to inhibit human IL-8 mediatedneutrophil chemotaxis.

FIGS. 59A–59B are graphs depicting the ability of PEG-succinimidemodified 6G4V11N35A F(ab′)₂ molecules to inhibit human IL-8 mediatedrelease of β-glucuronidase from neutrophils.

FIG. 60 is a graph depicting the theoretical molecular weight (dottedbars) and effective size (solid bars) of PEG-maleimide modified6G4V11N35A Fab′ molecules as determined by SEC-HPLC.

FIGS. 61A and 61B are SDS-PAGE gels depicting the electrophoreticmobility of various PEG-maleimide modified 6G4V11N35A Fab′ moleculesunder reducing and non-reducing conditions, respectively.

FIG. 62 contains size exclusion chromatograms (SEC-HPLC) depicting theretention times and effective (hydrodynamic) sizes of variousPEG-succinimide modified 6G4V11N35A F(ab′)₂ molecules.

FIG. 63 is a graph depicting the theoretical molecular weight (opencolumns), effective size determined by SEC-HPLC (solid columns), and theactual molecular weight determined by SEC-light scattering (shadedcolumns) for various PEG-succinimide modified 6G4V11N35A F(ab′)₂molecules.

FIG. 64 is an SDS-PAGE gel depicting the electrophoretic mobility ofvarious PEG-succinimide modified 6G4V11N35A F(ab′)₂ molecules. From leftto right, lane 1 contains unmodified F(ab′)₂, lane 2 contains F(ab′)₂coupled to two 40 kD branched PEG-succinimide molecules (denoted“Br(2)-40 kD(N)-F(ab′)2”), lane 3 contains F(ab′)₂ coupled to one 40 kDbranched PEG-succinimide molecule (denoted “Br(1)-40 kD-(N)-Fab′2”),lane 4 contains a mixture of F(ab′)₂ coupled to four 20 kD linearPEG-succinimide molecules and F(ab′)₂ coupled to five 20 kD linearPEG-succinimide molecules (denoted “L(4+5)-20 kD-(N)-Fab′2”), lane 5contains F(ab′)₂ coupled to one 20 kD linear PEG-succinimide molecule(denoted “L(1)-20 kD-(N)-Fab′2”), and lane 6 contains molecular weightstandards.

FIGS. 65A and 65B are graphs comparing the serum concentration vs. timeprofiles of various PEG-maleimide modified 6G4V11N35A Fab′ molecules(FIG. 65A) and various PEG-succinimide modified 6G4V11N35A F(ab′)₂molecules (FIG. 65B) in rabbits. In FIG. 65A, “bran.(1)40K(s)Fab′”denotes 6G4V11N35A Fab′ coupled to one 40 kD branched PEG-maleimidemolecule, “lin.(1)40K(s)Fab′” denotes 6G4V11N35A Fab′ coupled to one 40kD linear PEG-maleimide molecule, “lin.(1)30K(s)Fab′” denotes 6G4V11N35AFab′ coupled to one 30 kD linear PEG-maleimide molecule,“lin.(1)20K(s)Fab′” denotes 6G4V11N35A Fab′ coupled to one 20 kD linearPEG-maleimide molecule. In FIG. 65B, “bran.(2)40K(N)Fab′2” denotes6G4V11N35A F(ab′)₂ coupled to two 40 kD branched PEG-succinimidemolecules, “bran.(1)40K(N)Fab′2” denotes 6G4V11N35A F(ab′)₂ coupled toone 40 kD branched PEG-succinimide molecule, and “Fab′2” denotesunmodified 6G4V11N35A F(ab′)₂. In both FIGS. 65A and 65B, “IgG” denotesa full length IgG1 equivalent of the human-murine chimeric anti-rabbitIL-8 Fab described in Example F below.

FIG. 66 contains graphs comparing the serum concentration vs. timeprofiles of 6G4V11N35A Fab′ coupled to one 40 kD branched PEG-maleimidemolecule (denoted as “bran.(1)40K(s)Fab′”), 6G4V11N35A F(ab′)₂ coupledto one 40 kD branched PEG-succinimide molecule (denoted as“bran.(1)40K(N)Fab′2”), unmodified 6G4V11N35A F(ab′)₂ (denoted as“Fab′2”), unmodified 6G4V11N35A Fab′ (denoted as “Fab′”), and a fulllength IgG1 (denoted as “IgG”) equivalent of the human-murine chimericanti-rabbit IL-8 Fab described in Example F below.

FIG. 67 is a graph depicting the effect of 6G4V11N35A Fab′ coupled toone 40 kD branched PEG-maleimide molecule (denoted as “PEG 40 Kd”) andmurine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (full length IgG2a)(denoted as “6G4.2.5”) on gross weight of entire lung in an ARDS rabbitmodel.

FIG. 68 is a graph depicting the effect of 6G4V11N35A Fab′ coupled toone branched 40 kD PEG-maleimide molecule (denoted as “PEG 40 Kd”) andmurine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (full length IgG2a)(denoted as “6G4.2.5”) on BAL total leukocyte (light columns) andpolymorphonuclear cell (dark columns) counts in an ARDS rabbit model.Untreated (no therapeutics) control animal data is denoted as “Control”.

FIG. 69 is a graph depicting the effect of 6G4V11N35A Fab′ coupled toone branched 40 kD PEG-maleimide molecule (denoted as “PEG 40 Kd”) andmurine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (full length IgG2a)(denoted as “6G4.2.5”) on PaO2/FiO2 ratio at 24 hours-post treatment(light columns) and 48 hours post-treatment (dark columns) in an ARDSrabbit model. Untreated (no therapeutics) control animal data is denotedas “Control”.

FIG. 70A is a graph depicting PaO2/FiO2 ratios obtained in 100% oxygenat 24 hours after acid instillation for: (1) rabbits (n=5) treated with7 mg/1 kg IV 20 kD linear PEG-6G4V11N35E Fab′ at 10 minutes before and 6hours after acid instillation, (2) rabbits (n=7) treated with 5 mg/kg IVfull length IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 at10 minutes before acid instillation, (3) rabbits (n=3) treated with 5mg/kg IV 40 kD branched PEG-6G4V11N35A Fab′ at 10 minutes before acidinstillation, (4) rabbits (n=2) treated with 20 mg/kg IV 40 kD branchedPEG-6G4V11N35A Fab′ at 10 minutes before acid instillation, and (5)rabbits (n=25) treated with 5 ml IV saline at 10 minutes before and 6hours after acid instillation.

FIG. 70B is a graph depicting PaO2/FiO2 ratios obtained in 100% oxygenat 48 hours after acid instillation for: (1) rabbits (n=5) treated with7 mg/kg IV 20 kD linear PEG-6G4V11N35E Fab′ at 10 minutes before and 6hours after acid instillation, (2) rabbits (n=7) treated with 5 mg/kg IVfull length IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 at10 minutes before acid instillation, (3) rabbits (n=3) treated with 5mg/kg IV 40 kD branched PEG-6G4V11N35A Fab′ at 10 minutes before acidinstillation, (4) rabbits (n=2) treated with 20 mg/kg IV 40 kD branchedPEG-6G4V11N35A Fab′ at 10 minutes before acid instillation, and (5)rabbits (n=16) treated with 5 ml IV saline at 10 minutes before and 6hours after acid instillation.

FIG. 70C is a graph depicting gross lung weight (in grams)/body weight(in kilograms) ratios (denoted as “GLW/BW Ratio”) obtained at 72 hourspost reperfusion for: (1) rabbits (n=5) treated with 7 mg/kg IV 20 kDlinear PEG-6G4V11N35E Fab′ at 10 minutes before and 6 hours after acidinstillation, (2) rabbits (n=7) treated with 5 mg/kg IV full length IgGmurine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 at 10 minutes beforeacid instillation, (3) rabbits (n=3) treated with 5 mg/kg IV 40 kDbranched PEG-6G4V11N35A Fab′ at 10 minutes before acid instillation, (4)rabbits (n=3) treated with 20 mg/kg IV 40 kD branched PEG-6G4V11N35AFab′ at 10 minutes before acid instillation, and (5) rabbits (n=29)treated with 5 ml IV saline at 10 minutes before and 6 hours after acidinstillation.

FIG. 70D is a graph depicting total leukocyte (WBC) count in BAL fluid(represented in millions of cells counted in 20 ml BAL fluid) obtainedat 72 hours post reperfusion for: (1) rabbits (n=5) treated with 7 mg/kgIV 20 kD linear PEG-6G4V11N35E Fab′ at 10 minutes before and 6 hoursafter acid instillation, (2) rabbits (n=7) treated with 5 mg/kg IV fulllength IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 at 10minutes before acid instillation, (3) rabbits (n=3) treated with 5 mg/kgIV 40 kD branched PEG-6G4V11N35A Fab′ at 10 minutes before acidinstillation, (4) rabbits (n=3) treated with 20 mg/kg IV 40 kD branchedPEG-6G4V11N35A Fab′ at 10 minutes before acid instillation, and (5)rabbits (n=11) treated with 5 ml IV saline at 10 minutes before and 6hours after acid instillation.

FIG. 70E is a graph depicting total polymorphonuclear (PMN) cell countin BAL fluid (represented in millions of cells counted in 20 ml BALfluid) obtained at 72 hours post reperfusion for: (1) rabbits (n=5)treated with 7 mg/kg IV 20 kD linear PEG-6G4V11N35E Fab′ at 10 minutesbefore and 6 hours after acid instillation, (2) rabbits (n=7) treatedwith 5 mg/kg IV full length IgG murine anti-rabbit IL-8 monoclonalantibody 6G4.2.5 at 10 minutes before acid instillation, (3) rabbits(n=3) treated with 5 mg/kg IV 40 kD branched PEG-6G4V11N35A Fab′ at 10minutes before acid instillation, (4) rabbits (n=3) treated with 20mg/kg IV 40 kD branched PEG-6G4V11N35A Fab′ at 10 minutes before acidinstillation, and (5) rabbits (n=9) treated with 5 ml IV saline at 10minutes before and 6 hours after acid instillation.

FIG. 71 is a graph depicting the effect of pegylated anti-IL-8 Fab′ (asmeasured by percent change in ear volume at 1, 2 and 3 days postreperfusion) in a rabbit ear model of ischemia reperfusion injury. Thedata points from animals treated with empty vehicle (n=11), full lengthIgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (n=4), 20 kDlinear PEG-6G4V11N35E Fab′ (n=3), 30 kD linear PEG-6G4V11N35E Fab′(n=3), and 40 kD branched PEG-6G4V11N35E Fab′ (n=3) are denoted by openboxes, open diamonds, open circles, open triangles, and crossed boxes,respectively.

FIG. 72 is a graph comparing the serum concentration vs. time profilesof 20 kD linear PEG-maleimide modified Y0317 anti-human VEGF Fab′(denoted as “20K rhuMAb VEGF Fab IV”) and 40 kD branched PEG-maleimidemodified Y0317 anti-human VEGF Fab′ (denoted as “40K rhuMAb VEGF FabIV”) molecules administered intravenously in mice.

FIG. 73 is a graph comparing the serum concentration vs. time profilesof 20 kD linear PEG-maleimide modified Y0317 anti-human VEGF Fab′(denoted as “20K rhuMAb VEGF Fab IP”) and 40 kD branched PEG-maleimidemodified Y0317 anti-human VEGF Fab′ (denoted as “40K rhuMAb VEGF FabIP”) molecules administered intraperitoneally in mice.

FIG. 74 is a graph comparing inhibition of tumor growth in vivo in miceby intraperitoneal administration of 40 kD branched PEG-maleimidemodified Y0317 anti-human VEGF Fab′ (2 mg/kg loading dose on day 1followed by 0.9 mg/kg/day maintenance dose for the remainder of thestudy) (denoted “40K-LOW”), 40 kD branched PEG-maleimide modified Y0317anti-human VEGF Fab′ (6 mg/kg loading dose on day 1 followed by 2.7mg/kg/day maintenance dose for the remainder of the study) (denoted“40K-HIGH”), 40 kD branched PEG-6G4V11N35E Fab′ (6 mg/kg loading dose onday 1 followed by 2.7 mg/kg/day maintenance dose for the remainder ofthe study) (denoted “CNTRL FAB’), Y0317 anti-human VEGF MAb (8 mg/kgloading dose on day 1 followed by 0.8 mg/kg maintenance dose every thirdday for the remainder of the study) (denoted “2ND GEN AB”), andphosphate buffered saline at physiological pH (0.1 ml/day for theduration of the study) (denoted “PBS”).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims.

“Polymerase chain reaction” or “PCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued 28Jul. 1987. Generally, sequence information from the ends of the regionof interest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers can coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263 (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269–315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (k) and lambda (l), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ∈, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies) and antibody compositions with polyepitopic specificity.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety and (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety; andmultispecific or multivalent structures formed from antibody fragments.In an antibody fragment comprising one or more heavy chains, the heavychain(s) can contain any constant domain sequence (e.g. CH1 in the IgGisotype) found in a non-Fc region of an intact antibody, and/or cancontain any hinge region sequence found in an intact antibody, and/orcan contain a leucine zipper sequence fused to or situated in the hingeregion sequence or the constant domain sequence of the heavy chain(s).Suitable leucine zipper sequences include the jun and fos leucinezippers taught by Kostelney et al., J. Immunol., 148: 1547–1553 (1992)and the GCN4 leucine zipper described in the Examples below.

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), wherein the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.In the context of the foregoing definition, the term “structuredaggregate” refers to (1) any aggregate of molecules in aqueous solutionhaving a spheroid or spheroid shell structure, such that theheterogeneous molecule is not in a micelle or other emulsion structure,and is not anchored to a lipid bilayer, vesicle or liposome; and (2) anyaggregate of molecules in solid or insolubilized form, such as achromatography bead matrix, that does not release the heterogeneousmolecule into solution upon contact with an aqueous phase. Accordingly,the term “conjugate” as defined herein encompasses the aforementionedheterogeneous molecule in a precipitate, sediment, bioerodible matrix orother solid capable of releasing the heterogeneous molecule into aqueoussolution upon hydration of the solid.

Unless specifically indicated to the contrary, the terms “polymer”,“polymer molecule”, “nonproteinaceous polymer”, and “nonproteinaceouspolymer molecule” are used interchangeably and are defined as a moleculeformed by covalent linkage of two or more monomers, wherein none of themonomers is contained in the group consisting of alanine (Ala), cysteine(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe),glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine(Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine(Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val),tryptophan (Trp), and tyrosine (Tyr) residues.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each mAb is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they can be synthesized by hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabillyet al.). The “monoclonal antibodies” also include clones ofantigen-recognition and binding-site containing antibody fragments (Fvclones) isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624–628 (1991) and Marks etal., J. Mol. Biol., 222:581–597 (1991), for example.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-IL-8 antibody with a constant domain (e.g. “humanized”antibodies), or a light chain with a heavy chain, or a chain from onespecies with a chain from another species, or fusions with heterologousproteins, regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab′)₂,and Fv), so long as they exhibit the desired biological activity. (See,e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.; Mage and Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79–97(Marcel Dekker, Inc., New York, 1987).)

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂, or other antigen-binding subsequencesof antibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 321:522(1986); Reichmann et al., Nature 332:323 (1988); and Presta, Curr. Op.Struct. Biol. 2:593 (1992).

The terms “human vascular endothelial growth factor”, “vascularendothelial growth factor”, “human VEGF” and “VEGF” are usedinterchangeably herein to refer to the 165-amino acid human vascularendothelial cell growth factor polypeptide, and related 121-, 189-, and206-amino acid vascular endothelial cell growth factor polypeptides,described by Leung et al., Science 246:1306 (1989), and Houck et al.,Mol. Endocrin. 5:1806 (1991), together with the naturally occurringallelic and processed forms of such growth factor polypeptides.

The term “human VEGF receptor”, “VEGF receptor”, “human VEGFr” and“VEGFr” are used interchangeably herein to refer to a cellular receptorfor VEGF, ordinarily a cell-surface receptor found on vascularendothelial cells, as well as variants thereof which retain the abilityto bind human VEGF. One example of a VEGF receptor is the fms-liketyrosine kinase (flt), a transmembrane receptor in the tyrosine kinasefamily. DeVries et al., Science 255:989 (1992); Shibuya et al., Oncogene5:519 (1990). The flt receptor comprises an extracellular domain, atransmembrane domain, and an intracellular domain with tyrosine kinaseactivity. The extracellular domain is involved in the binding of VEGF,whereas the intracellular domain is involved in signal transduction.Another example of a VEGF receptor is the flk-1 receptor (also referredto as KDR). Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991);Terman et al., Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys.Res. Commun. 187:1579 (1992). Binding of VEGF to the flt receptorresults in the formation of at least two high molecular weightcomplexes, having apparent molecular weight of 205,000 and 300,000Daltons. The 300,000 Dalton complex is believed to be a dimer comprisingtwo receptor molecules bound to a single molecule of VEGF.

As used herein, the terms “human p185 receptor-like tyrosine kinase”,“c-Erb-B2”, “ErbB2”, “HER2”, and “HER2 receptor” are usedinterchangeably to refer to the c-Erb-B2 polypeptide described inYamamoto et al., Nature, 319: 230–234 (1986) (Genebank accession numberX03363).

As used herein, the terms “human CD20” and “CD20” refer to the B1cell-surface antigen (CD20) polypeptide described in Tedder et al.,Proc. Natl. Acad. Sci. (USA), 85: 208–212 (1988).

As used herein, the terms “human CD18” and “CD18” refer to the integrinβ-chain polypeptide (CD18) described in Kishimoto et al., Cell, 48:681–690 (1987).

As used herein, the terms “human CD11a” and “CD11a” refer to the humanCD11a polypeptide described in Edwards et al., J. Biol. Chem., 270:12635–12640 (1995), van Kooyk et al., J. Exp. Med., 183(3): 1247–1252(1996), or Champe et al., J. Biol. Chem., 270: 1388–1394 (1995).

As used herein, the terms “human IgE” and “IgE” refer to any humanimmunoglobulin of the E isotype or class that binds to the humanFc_(∈)RI receptor α-chain.

As used herein, the terms “human Fc_(∈)RI receptor α-chain”, “Fc_(∈)RIreceptor α-chain”, “human Fc_(∈)RI receptor”, “Fc_(∈)RI receptor”,“human Fc_(∈)RI”, and “Fc_(∈)RI” are used interchangeably to refer tothe human Fc_(∈)RI α-chain polypeptide described by Shimizu et al.,Proc. Natl. Acad. Sci. (USA), 85: 1907–1911 (1988).

As used herein, the terms “human Apo-2 receptor”, “Apo-2 receptor”,“human Apo-2”, and “Apo-2” are used interchangeably to refer to theApo-2 polypeptide described in FIG. 1 of WO 98/51793 (published Nov. 19,1998) (International Application No. PCT/US98/09704).

As used herein, the terms “human tumor necrosis factor-α”, “tumornecrosis factor-α”, “human TNF-α”, and “TNF-α” are used interchangeablyto refer to the human TNF-α polypeptide described in Pennica et al.,Nature, 512: 721 (1984) or in FIG. 10 of U.S. Pat. No. 4,650,674.

As used herein, the terms “human tissue factor” and “tissue factor” areused to refer to the human tissue factor polypeptide described in FIG. 2of European Patent No. 0 278 776 B1 (granted May 28, 1997).

As used herein, the terms “human α₄ integrin”, “α₄ integrin”, “humanα₄”, and “α₄” are used interchangeably to refer to the human VLA-4 α₄subunit polypeptide described in Takada et al., EMBO J., 8: 1361–1368(1989).

As used herein, the terms “human β₇ integrin”, “β₇ integrin”, “humanβ₇”, and “β₇” are used interchangeably to refer to the β₂-relatedintegrin polypeptide described in Yuan et al., International Immunology,2: 1097–1108 (1990).

As used herein, the terms “human GPIIIa integrin”, “GPIIIa integrin”,“human GPIIIa”, and “GPIIIa” are used interchangeably to refer to theGPIIIa polypeptide described in Fitzgerald et al., J. Biol. Chem.,262(9): 3936 (1987).

As used herein, the terms “human GPIIb integrin”, “GPIIb integrin”,“human GPIIb” and “GPIIb” are used interchangeably to refer to the GPIIbpolypeptide described in Fitzgerald et al., Biochem., 26: 8158 (1987).

As used herein, the terms “human GPIIb-IIIa integrin”, “GPIIb-IIIaintegrin”, “human GPIIb-IIIa”, and “GPIIb-IIIa” are used interchangeablyto refer to a GPIIb-GPIIIa integrin complex.

As used herein, the terms “human epidermal growth factor receptor”,“epidermal growth factor receptor”, “human EGFR”, and “EGFR” are usedinterchangeably to refer to the human epidermal growth factor receptorpolypeptide described in Ullrich et al., Nature, 309: 418–425 (1984).

As used herein, the terms “human CD3” and “CD3” are used interchangeablyto refer to the 20K T3 glycoprotein subunit of the human T-cell receptorcomplex described in van den Elsen et al., Nature, 312: 413–418 (1984).

As used herein, the terms “human interleukin-2 receptor α-chain”,“interleukin-2 receptor α-chain”, “human IL-2R α-chain”, “IL-2Rα-chain”, “human T-cell activation antigen”, “human TAC”, and “TAC” areused interchangeably to refer to the 272 amino acid interleukin-2receptor polypeptide described in Leonard et al., Nature, 311: 626–631(1984).

As used herein, the terms “anti-LFA-1 antibody”, “anti-LFA-1 monoclonalantibody” and “anti-LFA-1 MAb” refer to an antibody directed againsteither CD11a or CD18 or both. The anti-CD11a antibodies include, e.g.,MHM24 [Hildreth et al., Eur. J. Immunol., 13: 202–208 (1983)], R3.1(IgG1) [R. Rothlein, Boehringer Ingelheim Pharmaceuticals, Inc.,Ridgefield, Conn.], 25-3 (or 25.3), an IgG1 available from Immunotech,France [Olive et al., in Feldmann, ed., Human T cell Clones. A newApproach to Immune Regulation, Clifton, N.J., Humana, 1986 p. 173], KBA(IgG2a) [Nishimura et al., Cell. Immunol., 107: 32 (1987); Nishimura etal., ibid., 94: 122 (1985)], M7/15 (IgG2b) [Springer et al., Immunol.Rev., 68 171 (1982)], IOT16 [Vermot Desroches et al., Scand. J.Immunol., 33: 277–286 (1991)], SPVL7 [Vermot Desroches et al., supra],and M17 (IgG2a), available from ATCC, which are rat anti-murine CD11aantibodies.

Examples of anti-CD18 antibodies include MHM23 [Hildreth et al., supra],M18/2 (IgG2a) [Sanches-Madrid et al., J. Exp. Med., 158: 586 (1983)],H52 [Fekete et al., J. Clin. Lab Immunol., 31: 145–149 (1990)], Mas191c[Vermot Desroches et al., supra], IOT18 [Vermot Desroches et al.,supra], 60.3 [Taylor et al., Clin. Exp. Immunol., 71: 324–328 (1988)],and 60.1 [Campana et al., Eur. J. Immunol., 16: 537–542 (1986)].

The term “graft” as used herein refers to biological material derivedfrom a donor for transplantation into a recipient. Grafts include suchdiverse material as, for example, isolated cells such as islet cells,tissue such as the amniotic membrane of a newborn, bone marrow,hematopoietic precursor cells, and organs such as skin, heart, liver,spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g.,intestine, blood vessels, or esophagus), etc. The tubular organs can beused to replace damaged portions of esophagus, blood vessels, or bileduct. The skin grafts can be used not only for burns, but also as adressing to damaged intestine or to close certain defects such asdiaphragmatic hernia. The graft is derived from any mammalian source,including human, whether from cadavers or living donors. Preferably thegraft is bone marrow or an organ such as heart and the donor of thegraft and the host are matched for HLA class II antigens.

The term “donor” as used herein refers to the mammalian species, dead oralive, from which the graft is derived. Preferably, the donor is human.Human donors are preferably volunteer blood-related donors that arenormal on physical examination and of the same major ABO blood group,because crossing major blood group barriers possibly prejudices survivalof the allograft. It is, however, possible to transplant, for example, akidney of a type O donor into an A, B or AB recipient.

The term “transplant” and variations thereof refers to the insertion ofa graft into a host, whether the transplantation is syngeneic (where thedonor and recipient are genetically identical), allogeneic (where thedonor and recipient are of different genetic origins but of the samespecies), or xenogeneic (where the donor and recipient are fromdifferent species). Thus, in a typical scenario, the host is human andthe graft is an isograft, derived from a human of the same or differentgenetic origins. In another scenario, the graft is derived from aspecies different from that into which it is transplanted, such as ababoon heart transplanted into a human recipient host, and includinganimals from phylogenically widely separated species, for example, a pigheart valve, or animal beta islet cells or neuronal cells transplantedinto a human host.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

As used herein, protein, peptide and polypeptide are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers.

As used herein, the term “inflammatory disorders” refers to pathologicalstates resulting in inflammation, typically caused by neutrophilchemotaxis. Examples of such disorders include inflammatory skindiseases including psoriasis and atopic dermatitis; systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); ischemic reperfusiondisorders including surgical tissue reperfusion injury, myocardialischemic conditions such as myocardial infarction, cardiac arrest,reperfusion after cardiac surgery and constriction after percutaneoustransluminal coronary angioplasty, stroke, and abdominal aorticaneurysms; cerebral edema secondary to stroke; cranial trauma;hypovolemic shock; asphyxia; adult respiratory distress syndrome; acutelung injury; Behcet's Disease; dermatomyositis; polymyositis; multiplesclerosis; dermatitis; meningitis; encephalitis; uveitis;osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoidarthritis, Sjorgen's syndrome, vasculitis; diseases involving leukocytediapedesis; central nervous system (CNS) inflammatory disorder, multipleorgan injury syndrome secondary to septicaemia or trauma; alcoholichepatitis; bacterial pneumonia; antigen-antibody complex mediateddiseases including glomerulonephritis; sepsis; sarcoidosis;immunopathologic responses to tissue/organ transplantation;inflammations of the lung, including pleurisy, alveolitis, vasculitis,pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), andcystic fibrosis; etc. The preferred indications include acute lunginjury, adult respiratory distress syndrome, ischemic reperfusion(including surgical tissue reperfusion injury, myocardial ischemia, andacute myocardial infarction), hypovolemic shock, asthma, bacterialpneumonia and inflammatory bowel disease such as ulcerative colitis.

As used herein, the term “LFA-1-mediated disorder” refers topathological states caused by cell adherence interactions involving theLFA-1 receptor on lymphocytes. Examples of such disorders include T cellinflammatory responses such as inflammatory skin diseases includingpsoriasis; responses associated with inflammatory bowel disease (such asCrohn's disease and ulcerative colitis); adult respiratory distresssyndrome; dermatitis; meningitis; encephalitis; uveitis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses; skinhypersensitivity reactions (including poison ivy and poison oak);atherosclerosis; leukocyte adhesion deficiency; autoimmune diseases suchas rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetesmellitus, multiple sclerosis, Reynaud's syndrome, autoimmunethyroiditis, experimental autoimmune encephalomyelitis, Sjorgen'ssyndrome, juvenile onset diabetes, and immune responses associated withdelayed hypersensitivity mediated by cytokines and T-lymphocytestypically found in tuberculosis, sarcoidosis, polymyositis,granulomatosis and vasculitis; pernicious anemia; diseases involvingleukocyte diapedesis; CNS inflammatory disorder, multiple organ injurysyndrome secondary to septicaemia or trauma; autoimmune haemolyticanemia; myethemia gravis; antigen-antibody complex mediated diseases;all types of transplantations, including graft vs. host or host vs.graft disease; etc.

As used herein, the term “IgE-mediated disorder” means a condition ordisease which is characterized by the overproduction and/orhypersensitivity to the immunoglobulin IgE. Specifically it should beconstrued to include conditions associated with anaphylactichypersensitivity and atopic allergies, including for example: asthma,allergic rhinitis & conjunctivitis (hay fever), eczema, urticaria andfood allergies. However, the serious physiological condition ofanaphylactic shock, usually caused by bee or snake stings or parentalmedication is also encompassed under the scope of this term.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancer.

The terms “allergy” and “atopy” and all their grammatical variants areused synonymously herein to refer to any disease mediated by a Type I(Gell & Coombs classification) hypersensitivity reaction, includingallergic rhinitis, atopic dermatitis, anaphylaxis, allergic asthma.

As used herein, the terms “asthma”, “asthmatic disorder”, “asthmaticdisease”, and “bronchial asthma” refer to a condition of the lungs inwhich there is widespread narrowing of lower airways. “Atopic asthma”and “allergic asthma” refer to asthma that is a manifestation of anIgE-mediated hypersensitivity reaction in the lower airways, including,e.g., moderate or severe chronic asthma, such as conditions requiringthe frequent or constant use of inhaled or systemic steroids to controlthe asthma symptoms. A preferred indication is allergic asthma.

The term “allergic rhinitis” as used herein refers to anyallergen-induced nasal symptoms, including itching, sneezing, nasalcongestion, nasal discharge, and symptoms associated with nasal mucosalinflammation.

The terms “thrombotic disorder” and “prothrombotic disorder” as usedinterchangeably herein to refer to pathological conditions in which theblood coagulation cascade is activated (see, generally, Hoffbrand &Pettit, Essential Haematology, Blackwell Scientific Publications, Oxford(1980)). Such conditions include peripheral arterial obstruction, acutemyocardial infarction, deep vein thrombosis, pulmonary embolism,dissecting aneurysm, transient ischemic attack, restenosis, stroke andother occlusive disease or disorders such as unstable angina,disseminated intravascular coagulation, sepsis, surgical or infectiveshock, postoperative and post-delivery trauma, angioplasty,cardiopulmonary bypass and coronary bypass, incompatible bloodtransfusion, amotio placentae, thrombotic thrombocytopenic purpura,asthma, chronic or acute renal disease, diabetes, inflammations,atherosclerosis, hemolytic uremic syndrome, symmetric peripheralnecrosis, and allograft rejection in mammals including human.

The terms “hydrodynamic size”, “apparent size”, “apparent molecularweight”, “effective size” and “effective molecular weight” of a moleculeare used synonymously herein refer to the size of a molecule asdetermined by comparison to a standard curve produced with globularprotein molecular weight standards in a size exclusion chromatographysystem, wherein the standard curve is created by mapping the actualmolecular weight of each standard against its elution time observed inthe size exclusion chromatography system. Thus, the apparent size of atest molecule is derived by using the molecule's elution time toextrapolate a putative molecular weight from the standard curve.Preferably, the molecular weight standards used to create the standardcurve are selected such that the apparent size of the test moleculefalls within the linear portion of the standard curve.

II. Modes for Carrying Out the Invention

In one part, the invention arises from the surprising and unexpecteddiscovery that antibody fragment-polymer conjugates having an effectiveor apparent size significantly greater than the antibodyfragment-polymer conjugates described in the art confers an increase inserum half-life, an increase in mean residence time in circulation(MRT), and/or a decrease in serum clearance rate over underivatizedantibody fragment which far exceed the modest changes in such biologicalproperty or properties obtained with the art-known antibodyfragment-polymer conjugates. The present inventors have determined forthe first time that increasing the effective size of an antibodyfragment to at least about 500,000 D, or increasing the effective sizeof an antibody fragment by at least about 8 fold over the effective sizeof the parental antibody fragment, or derivatizing an antibody fragmentwith a polymer of at least about 20,000 D in molecular weight, yields amolecule with a commercially useful pharmacokinetic profile. The greatlyextended serum half-life, extended MRT, and/or reduced serum clearancerate of the conjugates of the invention makes such conjugates viablealternatives to intact antibodies used for therapeutic treatment of manydisease indications. Antibody fragments provide significant advantagesover intact antibodies, notably the fact that recombinant antibodyfragments can be made in bacterial cell expression systems. Bacterialcell expression systems provide several advantages over mammalian cellexpression systems, including reduced time and cost at both the researchand development and manufacturing stages of a product.

In another part, the present invention also arises from the humanizationof the 6G4.2.5 murine anti-rabbit IL-8 monoclonal antibody (“6G4.2.5”)described in WO 95/23865 (PCT/US95/02589 published Sep. 8, 1995), theentire disclosure of which is specifically incorporated herein byreference. The hybridoma producing antibody 6G4.2.5 was deposited onSep. 28, 1994 with the American Type Culture Collection and assignedATCC Accession No. HB 11722 as described in the Examples below. In oneaspect, the invention provides a humanized derivative of the 6G4.2.5antibody, variant 11 (referred to herein as “6G4.2.5v11”), in which themurine CDRs of 6G4.2.5 are grafted onto a consensus framework for humanlight chain κI and human IgG1 heavy chain subgroup III, followed byimporting three framework residues from the murine 6G4.2.5 parent heavychain variable domain sequence into analogous sites in the heavy chainvariable domain of the human template sequence, as described in theExamples below. In another aspect, the invention provides variants ofthe 6G4.2.5v11 antibody with certain amino acid substitution(s) yieldingincreased affinity for human IL-8 and/or promoting greater efficiency inrecombinant manufacturing processes.

It will be understood that in the context of this Section (II) and allsubsections thereof, every reference to “an antibody fragment” or “theantibody fragment” contained in a conjugate shall be a reference to oneor more antibody fragment(s) in the conjugate (consistent with thedefinition of the term “conjugate” set forth in Section (I) above),except where the number of antibody fragment(s) in the conjugate isexpressly indicated. It will be understood that in the context of thisSection (II) and all subsections thereof, every reference to “apolymer”, “a polymer molecule”, “the polymer”, or “the polymer molecule”contained in a conjugate shall be a reference to one or more polymermolecule(s) in the conjugate (consistent with the definition of the term“conjugate” set forth in Section (I) above), except where the number ofpolymer molecule(s) in the conjugate is expressly indicated.

1. Large Effective Size Antibody Fragment-Polymer Conjugates

In one aspect, the invention provides an antibody fragment covalentlyattached to a polymer to form a conjugate having an effective orapparent size of at least about 500,000 Daltons (D). In another aspect,the invention provides an antibody fragment covalently attached to apolymer to form a conjugate having an apparent size that is at leastabout 8 fold greater than the apparent size of the parental antibodyfragment. In yet another aspect, the invention provides an antibodyfragment covalently attached to a polymer of at least about 20,000 D inmolecular weight (MW). It will be appreciated that the unexpectedly andsurprisingly large increase in antibody fragment serum half-life,increase in MRT, and/or decrease in serum clearance rate can be achievedby using any type of polymer or number of polymer molecules which willprovide the conjugate with an effective size of at least about 500,000D, or by using any type of polymer or number of polymer molecules whichwill provide the conjugate with an effective size that is at least about8 fold greater than the effective size of the parental antibodyfragment, or by using any type or number of polymers wherein eachpolymer molecule is at least about 20,000 D in MW. Thus, the inventionis not dependent on the use of any particular polymer or molar ratio ofpolymer to antibody fragment in the conjugate.

In addition, the beneficial aspects of the invention extend to antibodyfragments without regard to antigen specificity. Although variationsfrom antibody to antibody are to be expected, the antigen specificity ofa given antibody will not substantially impair the extraordinaryimprovement in serum half-life, MRT, and/or serum clearance rate forantibody fragments thereof that can be obtained by derivatizing theantibody fragments as taught herein. The invention can be applied to anantibody fragment specific for any antigen of interest, including, e.g.,renin; a growth hormone, including human growth hormone and bovinegrowth hormone; growth hormone releasing factor; parathyroid hormone;thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; follicle stimulating hormone;calcitonin; luteinizing hormone; glucagon; clotting factors such asfactor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or human urine ortissue-type plasminogen activator (t-PA); bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha and -beta;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha); aserum albumin such as human serum albumin; Muellerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asbeta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen(CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growthfactor (VEGF); receptors for hormones or growth factors; protein A or D;rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe HIV envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

In one embodiment, the conjugate has an effective size of at least about500,000 D, or at least about 800,000 D, or at least about 900,000 D, orat least about 1,000,000 D, or at least about 1,200,000 D, or at leastabout 1,400,000 D, or at least about 1,500,000 D, or at least about1,800,000 D, or at least about 2,000,000 D, or at least about 2,500,000D.

In another embodiment, the conjugate has an effective size of at orabout 500,000 D to at or about 10,000,000 D, or an effective size of ator about 500,000 D to at or about 8,000,000 D, or an effective size ofat or about 500,000 D to at or about 5,000,000 D, or an effective sizeof at or about 500,000 D to at or about 4,000,000 D, or an effectivesize of at or about 500,000 D to at or about 3,000,000 D, or aneffective size of at or about 500,000 D to at or about 2,500,000 D, oran effective size of at or about 500,000 D to at or about 2,000,000. D,or an effective size of at or about 500,000 D to at or about 1,800,000D, or an effective size of at or about 500,000 D to at or about1,600,000 D, or an effective size of at or about 500,000 D to at orabout 1,500,000 D, or an effective size of at or about 500,000 D to ator about 1,000,000 D.

In another embodiment, the conjugate has an effective size of at orabout 800,000 D to at or about 10,000,000 D, or an effective size of ator about 800,000 D to at or about 8,000,000 D, or an effective size ofat or about 800,000 D to at or about 5,000,000 D, or an effective sizeof at or about 800,000 D to at or about 4,000,000 D, or an effectivesize of at or about 800,000 D to at or about 3,000,000 D, or aneffective size of at or about 800,000 D to at or about 2,500,000 D, oran effective size of at or about 800,000 D to at or about 2,000,000 D,or an effective size of at or about 800,000 D to at or about 1,800,000D, or an effective size of at or about 800,000 D to at or about1,600,000 D, or an effective size of at or about 800,000 D to at orabout 1,500,000 D, or an effective size of at or about 800,000 D to ator about 1,000,000 D.

In another embodiment, the conjugate has an effective size of at orabout 900,000 D to at or about 10,000,000 D, or an effective size of ator about 900,000 D to at or about 8,000,000 D, or an effective size ofat or about 900,000 D to at or about 5,000,000 D, or an effective sizeof at or about 900,000 D to at or about 4,000,000 D, or an effectivesize of at or about 900,000 D to at or about 3,000,000 D, or aneffective size of at or about 900,000 D to at or about 2,500,000 D, oran effective size of at or about 900,000 D to at or about 2,000,000 D,or an effective size of at or about 900,000 D to at or about 1,800,000D, or an effective size of at or about 900,000 D to at or about1,600,000 D, or an effective size of at or about 900,000 D to at orabout 1,500,000 D.

In another embodiment, the conjugate has an effective size of at orabout 1,000,000 D to at or about 10,000,000 D, or an effective size ofat or about 1,000,000 D to at or about 8,000,000 D, or an effective sizeof at or about 1,000,000 D to at or about 5,000,000 D, or an effectivesize of at or about 1,000,000 D to at or about 4,000,000 D, or aneffective size of at or about 1,000,000 D to at or about 3,000,000 D, oran effective size of at or about 1,000,000 D to at or about 2,500,000 D,or an effective size of at or about 1,000,000 D to at or about 2,000,000D, or an effective size of at or about 1,000,000 D to at or about1,800,000 D, or an effective size of at or about 1,000,000 D to at orabout 1,600,000 D, or an effective size of at or about 1,000,000 D to ator about 1,500,000 D.

In a further embodiment, the conjugate has an effective size that is atleast about 8 fold greater, or at least about 10 fold greater, or atleast about 12 fold greater, or at least about 15 fold greater, or atleast about 18 fold greater, or at least about 20 fold greater, or atleast about 25 fold greater, or at least about 28 fold greater, or atleast about 30 fold greater, or at least about 40 fold greater, than theeffective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about8 fold to about 100 fold greater, or is about 8 fold to about 80 foldgreater, or is about 8 fold to about 50 fold greater, or is about 8 foldto about 40 fold greater, or is about 8 fold to about 30 fold greater,or is about 8 fold to about 28 fold greater, or is about 8 fold to about25 fold greater, or is about 8 fold to about 20 fold greater, or isabout 8 fold to about 18 fold greater, or is about 8 fold to about 15fold greater, than the effective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about12 fold to about 100 fold greater, or is about 12 fold to about 80 foldgreater, or is about 12 fold to about 50 fold greater, or is about 12fold to about 40 fold greater, or is about 12 fold to about 30 foldgreater, or is about 12 fold to about 28 fold greater, or is about 12fold to about 25 fold greater, or is about 12 fold to about 20 foldgreater, or is about 12 fold to about 18 fold greater, or is about 12fold to about 15 fold greater, than the effective size of the parentalantibody fragment.

In another embodiment, the conjugate has an effective size that is about15 fold to about 100 fold greater, or is about 15 fold to about 80 foldgreater, or is about 15 fold to about 50 fold greater, or is about 15fold to about 40 fold greater, or is about 15 fold to about 30 foldgreater, or is about 15 fold to about 28 fold greater, or is about 15fold to about 25 fold greater, or is about 15 fold to about 20 foldgreater, or is about 15 fold to about 18 fold greater, than theeffective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about18 fold to about 100 fold greater, or is about 18 fold to about 80 foldgreater, or is about 18 fold to about 50 fold greater, or is about 18fold to about 40 fold greater, or is about 18 fold to about 30 foldgreater, or is about 18 fold to about 28 fold greater, or is about 18fold to about 25 fold greater, or is about 18 fold to about 20 foldgreater, than the effective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about20 fold to about 100 fold greater, or is about 20 fold to about 80 foldgreater, or is about 20 fold to about 50 fold greater, or is about 20fold to about 40 fold greater, or is about 20 fold to about 30 foldgreater, or is about 20 fold to about 28 fold greater, or is about 20fold to about 25 fold greater, than the effective size of the parentalantibody fragment.

In another embodiment, the conjugate has an effective size that is about25 fold to about 100 fold greater, or is about 25 fold to about 80 foldgreater, or is about 25 fold to about 50 fold greater, or is about 25fold to about 40 fold greater, or is about 25 fold to about 30 foldgreater, or is about 25 fold to about 28 fold greater, than theeffective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about28 fold to about 100 fold greater, or is about 28 fold to about 80 foldgreater, or is about 28 fold to about 50 fold greater, or is about 28fold to about 40 fold greater, or is about 28 fold to about 30 foldgreater, than the effective size of the parental antibody fragment.

In another embodiment, the conjugate has an effective size that is about30 fold to about 100 fold greater, or is about 30 fold to about 80 foldgreater, or is about 30 fold to about 50 fold greater, or is about 30fold to about 40 fold greater, than the effective size of the parentalantibody fragment.

In another embodiment, the conjugate has an effective size that is about40 fold to about 100 fold greater, or is about 40 fold to about 80 foldgreater, or is about 40 fold to about 50 fold greater, than theeffective size of the parental antibody fragment.

In still another embodiment, the conjugate is an antibody fragmentcovalently attached to at least one polymer having an actual MW of atleast about 20,000 D.

In a further embodiment, the conjugate is an antibody fragmentcovalently attached to at least one polymer having an actual MW of atleast about 30,000 D.

In yet another embodiment, the conjugate is an antibody fragmentcovalently attached to at least one polymer having an actual MW of atleast about 40,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one polymer having an actual MW that is at or about20,000 D to at or about 300,000 D, or is at or about 30,000 D to at orabout 300,000 D, or is at or about 40,000 D to at or about 300,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one polymer having an actual MW that is at or about20,000 D to at or about 100,000 D, or is at or about 30,000 D to at orabout 100,000 D, or is at or about 40,000 D to at or about 100,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one polymer having an actual MW that is at or about20,000 D to at or about 70,000 D, or is at or about 30,000 D to at orabout 70,000 D, or is at or about 40,000 D to at or about 70,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one polymer having an actual MW that is at or about20,000 D to at or about 50,000 D, or is at or about 30,000 D to at orabout 50,000 D, or is at or about 40,000 D to at or about 50,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one polymer having an actual MW that is at or about20,000 D to at or about 40,000 D, or is at or about 30,000 D to at orabout 40,000 D.

The conjugates of the invention can be made using any suitable techniquenow known or hereafter developed for derivatizing antibody fragmentswith polymers. It will be appreciated that the invention is not limitedto conjugates utilizing any particular type of linkage between anantibody fragment and a polymer.

The conjugates of the invention include species wherein a polymer iscovalently attached to a non-specific site or non-specific sites on theparental antibody fragment, i.e. polymer attachment is not targeted to aparticular region or a particular amino acid residue in the parentalantibody fragment. In such embodiments, the coupling chemistry can, forexample, utilize the free epsilon amino groups of lysine residues in theparental antibody as attachment sites for the polymer, wherein suchlysine residue amino groups are randomly derivatized with polymer.

In addition, the conjugates of the invention include species wherein apolymer is covalently attached to a specific site or specific sites onthe parental antibody fragment, i.e. polymer attachment is targeted to aparticular region or a particular amino acid residue or residues in theparental antibody fragment. In such embodiments, the coupling chemistrycan, for example, utilize the free sulfhydryl group of a cysteineresidue not in a disulfide bridge in the parental antibody fragment. Inone embodiment, one or more cysteine residue(s) is (are) engineered intoa selected site or sites in the parental antibody fragment for thepurpose of providing a specific attachment site or sites for polymer.The polymer can be activated with any functional group that is capableof reacting specifically with the free sulfhydryl or thiol group(s) onthe parental antibody, such as maleimide, sulfhydryl, thiol, triflate,tesylate, aziridine, exirane, and 5-pyridyl functional groups. Thepolymer can be coupled to the parental antibody fragment using anyprotocol suitable for the chemistry of the coupling system selected,such as the protocols and systems described in Section (II)(1)(b) or inSection (T) of the Examples below.

In another embodiment, polymer attachment is targeted to the hingeregion of the parental antibody fragment. The location of the hingeregion varies according to the isotype of the parental antibody.Typically, the hinge region of IgG, IgD and IgA isotype heavy chains iscontained in a proline rich peptide sequence extending between theC_(H)1 and C_(H)2 domains. In a preferred embodiment, a cysteine residueor residues is (are) engineered into the hinge region of the parentalantibody fragment in order to couple polymer specifically to a selectedlocation in the hinge region.

In one aspect, the invention encompasses a conjugate having any molarratio of polymer to antibody fragment that endows the conjugate with anapparent size in the desired range as taught herein. The apparent sizeof the conjugate will depend in part upon the size and shape of thepolymer used, the size and shape of the antibody fragment used, thenumber of polymer molecules attached to the antibody fragment, and thelocation of such attachment site(s) on the antibody fragment. Theseparameters can easily be identified and maximized to obtain the aconjugate with the desired apparent size for any type of antibodyfragment, polymer and linkage system.

In another aspect, the invention encompasses a conjugate with a polymerto antibody fragment molar ratio of no more than about 10:1, or no morethan about 5:1, or no more than about 4:1, or no more than about 3:1, orno more than about 2:1, or no more than 1:1.

In yet another aspect, the invention encompasses a conjugate wherein theantibody fragment is attached to about 10 or fewer polymer molecules,each polymer molecule having a molecular weight of at least about 20,000D, or at least about 30,000 D, or at least about 40,000 D. In anotherembodiment, the conjugate contains an antibody fragment attached toabout 5 or fewer polymer molecules, each polymer molecule having amolecular weight of at least about 20,000 D, or at least about 30,000 D,or at least about 40,000 D. In still another embodiment, the conjugatecontains an antibody fragment attached to about 4 or fewer polymermolecules, each polymer molecule having a molecular weight of at leastabout 20,000 D, or at least about 30,000 D, or at least about 40,000 D.In a further embodiment, the conjugate contains an antibody fragmentattached to about 3 or fewer polymer molecules, each polymer moleculehaving a molecular weight of at least about 20,000 D, or at least about30,000 D, or at least about 40,000 D. In an additional embodiment, theconjugate contains an antibody fragment attached to about 2 or fewerpolymer molecules, each polymer molecule having a molecular weight of atleast about 20,000 D, or at least about 30,000 D, or at least about40,000 D. Also provided herein is a conjugate containing an antibodyfragment attached to a single polymer molecule having a molecular weightof at least about 20,000 D, or at least about 30,000 D, or at leastabout 40,000 D.

In still another aspect, the invention encompasses a conjugate whereinevery polymer molecule in the conjugate has a molecular weight that isat or about 20,000 D to at or about 300,000 D, or is at or about 30,000D to at or about 300,000 D, or is at or about 40,000 D to at or about300,000 D, and wherein the conjugate contains no more than about 10polymer molecules, or no more than about 5 polymer molecules, or no morethan about 4 polymer molecules, or no more than about 3 polymermolecules, or no more than about 2 polymer molecules, or no more than 1polymer molecule.

In still another aspect, the invention encompasses a conjugate whereinevery polymer molecule in the conjugate has a molecular weight that isat or about 20,000 D to at or about 100,000 D, or is at or about 30,000D to at or about 100,000 D, or is at or about 40,000 D to at or about100,000 D, and wherein the conjugate contains no more than about 10polymer molecules, or no more than about 5 polymer molecules, or no morethan about 4 polymer molecules, or no more than about 3 polymermolecules, or no more than about 2 polymer molecules, or no more than 1polymer molecule.

In still another aspect, the invention encompasses a conjugate whereinevery polymer molecule in the conjugate has a molecular weight that isat or about 20,000 D to at or about 70,000 D, or is at or about 30,000 Dto at or about 70,000 D, or is at or about 40,000 D to at or about70,000 D, and wherein the conjugate contains no more than about 10polymer molecules, or no more than about 5 polymer molecules, or no morethan about 4 polymer molecules, or no more than about 3 polymermolecules, or no more than about 2 polymer molecules, or no more than 1polymer molecule.

In still another aspect, the invention encompasses a conjugate whereinevery polymer molecule in the conjugate has a molecular weight that isat or about 20,000 D to at or about 50,000 D, or is at or about 30,000 Dto at or about 50,000 D, or is at or about 40,000 D to at or about50,000 D, and wherein the conjugate contains no more than about 10polymer molecules, or no more than about 5 polymer molecules, or no morethan about 4 polymer molecules, or no more than about 3 polymermolecules, or no more than about 2 polymer molecules, or no more than 1polymer molecule.

In still another aspect, the invention encompasses a conjugate whereinevery polymer molecule in the conjugate has a molecular weight that isat or about 20,000 D to at or about 40,000 D, or is at or about 30,000 Dto at or about 40,000 D, and wherein the conjugate contains no more thanabout 10 polymer molecules, or no more than about 5 polymer molecules,or no more than about 4 polymer molecules, or no more than about 3polymer molecules, or no more than about 2 polymer molecules, or no morethan 1 polymer molecule.

It is believed that the serum half-life, MRT and/or serum clearance rateof any antibody fragment can be greatly improved by derivatizing theantibody fragment with polymer as taught herein. In one embodiment, theconjugate contains an antibody fragment selected from the groupconsisting of Fab, Fab′, Fab′-SH, Fv, scFv and F(ab′)₂.

In a preferred embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinevery polymer molecule in the conjugate is attached to the hinge regionof the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,every polymer molecule in the conjugate molecule is attached to thehinge region of the antibody fragment, and the conjugate contains nomore than about 10 polymer molecules, or no more than about 5 polymermolecules, or no more than about 4 polymer molecules, or no more thanabout 3 polymer molecules, or no more than about 2 polymer molecules, orno more than 1 polymer molecule.

In yet another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment attached to no more than about 2 polymer molecules,wherein every polymer molecule is attached to a cysteine residue in thelight or heavy chain of the antibody fragment that would ordinarily formthe disulfide bridge linking the light and heavy chains, wherein thedisulfide bridge is avoided by substituting another amino acid, such asserine, for the corresponding cysteine residue in the opposite chain.

In a further embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule andthe polymer is coupled to a cysteine residue in the light or heavy chainof the antibody fragment that would ordinarily form the disulfide bridgelinking the light and heavy chains, wherein the disulfide bridge isavoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In an additional embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at least about 20,000 D inmolecular weight, or at least about 30,000 in molecular weight, or atleast about 40,000 D in molecular weight, every polymer molecule in theconjugate is attached to the hinge region of the antibody fragment, andthe conjugate contains no more than about 10 polymer molecules, or nomore than about 5 polymer molecules, or no more than about 4 polymermolecules, or no more than about 3 polymer molecules, or no more thanabout 2 polymer molecules, or no more than 1 polymer molecule.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at or about 20,000 D to at or about300,000 D in molecular weight, or is at or about 30,000 D to at or about300,000 D in molecular weight, or is at or about 40,000 D to at or about300,000 D in molecular weight, every polymer molecule in the conjugateis attached to the hinge region of the antibody fragment, and theconjugate contains no more than about 10 polymer molecules, or no morethan about 5 polymer molecules, or no more than about 4 polymermolecules, or no more than about 3 polymer molecules, or no more thanabout 2 polymer molecules, or no more than 1 polymer molecule.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at or about 20,000 D to at or about100,000 D in molecular weight, or is at or about 30,000 D to at or about100,000 D in molecular weight, or is at or about 40,000 D to at or about100,000 D in molecular weight, every polymer molecule in the conjugateis attached to the hinge region of the antibody fragment, and theconjugate contains no more than about 10 polymer molecules, or no morethan about 5 polymer molecules, or no more than about 4 polymermolecules, or no more than about 3 polymer molecules, or no more thanabout 2 polymer molecules, or no more than 1 polymer molecule.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at or about 20,000 D to at or about70,000 D in molecular weight, or is at or about 30,000 D to at or about70,000 D in molecular weight, or is at or about 40,000 D to at or about70,000 D in molecular weight, every polymer molecule in the conjugate isattached to the hinge region of the antibody fragment, and the conjugatecontains no more than about 10 polymer molecules, or no more than about5 polymer molecules, or no more than about 4 polymer molecules, or nomore than about 3 polymer molecules, or no more than about 2 polymermolecules, or no more than 1 polymer molecule.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at or about 20,000 D to at or about50,000 D in molecular weight, or is at or about 30,000 D to at or about50,000 D in molecular weight, or is at or about 40,000 D to at or about50,000 D in molecular weight, every polymer molecule in the conjugate isattached to the hinge region of the antibody fragment, and the conjugatecontains no more than about 10 polymer molecules, or no more than about5 polymer molecules, or no more than about 4 polymer molecules, or nomore than about 3 polymer molecules, or no more than about 2 polymermolecules, or no more than 1 polymer molecule.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, everypolymer molecule in the conjugate is at or about 20,000 D to at or about40,000 D in molecular weight, or is at or about 30,000 D to at or about40,000 D in molecular weight, every polymer molecule in the conjugate isattached to the hinge region of the antibody fragment, and the conjugatecontains no more than about 10 polymer molecules, or no more than about5 polymer molecules, or no more than about 4 polymer molecules, or nomore than about 3 polymer molecules, or no more than about 2 polymermolecules, or no more than 1 polymer molecule.

In a further embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at least about 20,000 D inmolecular weight, or at least about 30,000 D in molecular weight, or atleast about 40,000 D in molecular weight, and wherein every polymermolecule in the conjugate is attached to a cysteine residue in the lightor heavy chain of the antibody fragment that would ordinarily form thedisulfide bridge linking the light and heavy chains, wherein thedisulfide bridge is avoided by substituting another amino acid, such asserine, for the corresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at or about 20,000 D to at orabout 300,000 D in molecular weight, or is at or about 30,000 D to at orabout 300,000 D in molecular weight, or is at or about 40,000 D to at orabout 300,000 D in molecular weight, and wherein every polymer moleculein the conjugate is attached to a cysteine residue in the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains, wherein the disulfide bridgeis avoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at or about 20,000 D to at orabout 100,000 D in molecular weight, or is at or about 30,000 D to at orabout 100,000 D in molecular weight, or is at or about 40,000 D to at orabout 100,000 D in molecular weight, and wherein every polymer moleculein the conjugate is attached to a cysteine residue in the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains, wherein the disulfide bridgeis avoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at or about 20,000 D to at orabout 70,000 D in molecular weight, or is at or about 30,000 D to at orabout 70,000 D in molecular weight, or is at or about 40,000 D to at orabout 70,000 D in molecular weight, and wherein every polymer moleculein the conjugate is attached to a cysteine residue in the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains, wherein the disulfide bridgeis avoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at or about 20,000 D to at orabout 50,000 D in molecular weight, or is at or about 30,000 D to at orabout 50,000 D in molecular weight, or is at or about 40,000 D to at orabout 50,000 D in molecular weight, and wherein every polymer moleculein the conjugate is attached to a cysteine residue in the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains, wherein the disulfide bridgeis avoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains a F(ab′)₂ antibodyfragment attached to no more than about 2 polymer molecules, whereinevery polymer molecule in the conjugate is at or about 20,000 D to at orabout 40,000 D in molecular weight, or is at or about 30,000 D to at orabout 40,000 D in molecular weight, and wherein every polymer moleculein the conjugate is attached to a cysteine residue in the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains, wherein the disulfide bridgeis avoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In yet another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at least about 20,000 D in molecularweight, or at least about 30,000 D in molecular weight, or at leastabout 40,000 D in molecular weight, wherein the polymer molecule isattached to a cysteine residue in the light or heavy chain of theantibody fragment that would ordinarily form the disulfide bridgelinking the light and heavy chains, wherein the disulfide bridge isavoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about300,000 D in molecular weight, or is at or about 30,000 D to at or about300,000 D in molecular weight, or is at or about 40,000 D to at or about300,000 D in molecular weight, wherein the polymer molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about100,000 D in molecular weight, or is at or about 30,000 D to at or about100,000 D in molecular weight, or is at or about 40,000 D to at or about100,000 D in molecular weight, wherein the polymer molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about70,000 D in molecular weight, or is at or about 30,000 D to at or about70,000 D in molecular weight, or is at or about 40,000 D to at or about70,000 D in molecular weight, wherein the polymer molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about50,000 D in molecular weight, or is at or about 30,000 D to at or about50,000 D in molecular weight, or is at or about 40,000 D to at or about50,000 D in molecular weight, wherein the polymer molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about40,000 D in molecular weight, or is at or about 30,000 D to at or about40,000 D in molecular weight, wherein the polymer molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In still another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at least about 20,000 D in molecularweight, or at least about 30,000 D in molecular weight, or at leastabout 40,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about300,000 D in molecular weight, or is at or about 30,000 D to at or about300,000 D in molecular weight, or is at or about 40,000 D to at or about300,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about100,000 D in molecular weight, or is at or about 30,000 D to at or about100,000 D in molecular weight, or is at or about 40,000 D to at or about100,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about70,000 D in molecular weight, or is at or about 30,000 D to at or about70,000 D in molecular weight, or is at or about 40,000 D to at or about70,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about50,000 D in molecular weight, or is at or about 30,000 D to at or about50,000 D in molecular weight, or is at or about 40,000 D to at or about50,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

In another embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 polymer molecule,wherein the polymer molecule is at or about 20,000 D to at or about40,000 D in molecular weight, or is at or about 30,000 D to at or about40,000 D in molecular weight, and wherein the polymer molecule isattached to the hinge region of the antibody fragment.

Although any type of polymer is contemplated for use in constructing theconjugates of the invention, including the polymers and chemical linkagesystems described in Section (II)(1)(b) below, polyethylene glycol (PEG)polymers are preferred for use herein.

In one embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW of at least about20,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW of at least about30,000 D.

In yet another embodiment, the conjugate is an antibody fragmentcovalently attached to at least one PEG having an actual MW of at leastabout 40,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW that is at or about20,000 D to at or about 300,000 D, or is at or about 30,000 D to at orabout 300,000 D, or is at or about 40,000 D to at or about 300,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW that is at or about20,000 D to at or about 100,000 D, or is at or about 30,000 D to at orabout 100,000 D, or is at or about 40,000 D to at or about 100,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW that is at or about20,000 D to at or about 70,000 D, or is at or about 30,000 D to at orabout 70,000 D, or is at or about 40,000 D to at or about 70,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW that is at or about20,000 D to at or about 50,000 D, or is at or about 30,000 D to at orabout 50,000 D, or is at or about 40,000 D to at or about 50,000 D.

In another embodiment, the conjugate is an antibody fragment covalentlyattached to at least one PEG having an actual MW that is at or about20,000 D to at or about 40,000 D, or is at or about 30,000 D to at orabout 40,000 D.

In another aspect, the invention encompasses a conjugate with a PEG toantibody fragment molar ratio of no more than about 10:1, or no morethan about 5:1, or no more than about 4:1, or no more than about 3:1, orno more than about 2:1, or no more than 1:1.

In yet another aspect, the invention encompasses a conjugate wherein theantibody fragment is attached to about 10 or fewer PEG molecules, eachPEG molecule having a molecular weight of at least about 20,000 D, or atleast about 30,000 D, or at least about 40,000 D. In another embodiment,the conjugate contains an antibody fragment attached to about 5 or fewerPEG molecules, each PEG molecule having a molecular weight of at leastabout 20,000 D, or at least about 30,000 D, or at least about 40,000 D.In still another embodiment, the conjugate contains an antibody fragmentattached to about 4 or fewer PEG molecules, each PEG molecule having amolecular weight of at least about 20,000 D, or at least about 30,000 D,or at least about 40,000 D. In a further embodiment, the conjugatecontains an antibody fragment attached to about 3 or fewer PEGmolecules, each PEG molecule having a molecular weight of at least about20,000 D, or at least about 30,000 D, or at least about 40,000 D. In anadditional embodiment, the conjugate contains an antibody fragmentattached to about 2 or fewer PEG molecules, each PEG molecule having amolecular weight of at least about 20,000 D, or at least about 30,000 D,or at least about 40,000 D. Also provided herein is a conjugatecontaining an antibody fragment attached to a single PEG molecule havinga molecular weight of at least about 20,000 D, or at least about 30,000D, or at least about 40,000 D.

In another aspect, the invention encompasses a conjugate wherein theantibody fragment is derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 300,000 D inmolecular weight, or is at or about 30,000 D to at or about 300,000 D inmolecular weight, or is at or about 40,000 D to at or about 300,000 D inmolecular weight, and wherein the conjugate contains no more than about10 PEG molecules, or no more than about 5 PEG molecules, or no more thanabout 4 PEG molecules, or no more than about 3 PEG molecules, or no morethan about 2 PEG molecules, or no more than 1 PEG molecule.

In another aspect, the invention encompasses a conjugate wherein theantibody fragment is derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 100,000 D inmolecular weight, or is at or about 30,000 D to at or about 100,000 D inmolecular weight, or is at or about 40,000 D to at or about 100,000 D inmolecular weight, and wherein the conjugate contains no more than about10 PEG molecules, or no more than about 5 PEG molecules, or no more thanabout 4 PEG molecules, or no more than about 3 PEG molecules, or no morethan about 2 PEG molecules, or no more than 1 PEG molecule.

In another aspect, the invention encompasses a conjugate wherein theantibody fragment is derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 70,000 D inmolecular weight, or is at or about 30,000 D to at or about 70,000 D inmolecular weight, or is at or about 40,000 D to at or about 70,000 D inmolecular weight, and wherein the conjugate contains no more than about10 PEG molecules, or no more than about 5 PEG molecules, or no more thanabout 4 PEG molecules, or no more than about 3 PEG molecules, or no morethan about 2 PEG molecules, or no more than 1 PEG molecule.

In another aspect, the invention encompasses a conjugate wherein theantibody fragment is derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 50,000 D inmolecular weight, or is at or about 30,000 D to at or about 50,000 D inmolecular weight, or is at or about 40,000 D to at or about 50,000 D inmolecular weight, and wherein the conjugate contains no more than about10 PEG molecules, or no more than about 5 PEG molecules, or no more thanabout 4 PEG molecules, or no more than about 3 PEG molecules, or no morethan about 2 PEG molecules, or no more than 1 PEG molecule.

In another aspect, the invention encompasses a conjugate wherein theantibody fragment is derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 40,000 D inmolecular weight, or is at or about 30,000 D to at or about 40,000 D inmolecular weight, and wherein the conjugate contains no more than about10 PEG molecules, or no more than about 5 PEG molecules, or no more thanabout 4 PEG molecules, or no more than about 3 PEG molecules, or no morethan about 2 PEG molecules, or no more than 1 PEG molecule.

In still another aspect, the invention encompasses a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, Fab′-SH and F(ab′)₂, wherein the antibody fragment isattached to about 10 or fewer PEG molecules, each PEG molecule having amolecular weight of at least about 20,000 D, or at least about 30,000 D,or at least about 40,000 D. In another embodiment, the foregoingconjugate contains an antibody fragment attached to about 5 or fewer PEGmolecules, each PEG molecule having a molecular weight of at least about20,000 D, or at least about 30,000 D, or at least about 40,000 D. Instill another embodiment, the foregoing conjugate contains an antibodyfragment attached to about 4 or fewer PEG molecules, each PEG moleculehaving a molecular weight of at least about 20,000 D, or at least about30,000 D, or at least about 40,000 D. In a further embodiment, theforegoing conjugate contains an antibody fragment attached to about 3 orfewer PEG molecules, each PEG molecule having a molecular weight of atleast about 20,000 D, or at least about 30,000 D, or at least about40,000 D. In an additional embodiment, the foregoing conjugate containsan antibody fragment attached to about 2 or fewer PEG molecules, eachPEG molecule having a molecular weight of at least about 20,000 D, or atleast about 30,000 D, or at least about 40,000 D. Also provided hereinis the foregoing conjugate that contains an antibody fragment attachedto a single PEG molecule having a molecular weight of at least about20,000 D, or at least about 30,000 D, or at least about 40,000 D.

In another aspect, the invention encompasses a conjugate containing anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH and F(ab′)₂, wherein the antibody fragment is derivatized withPEG, wherein every PEG molecule in the conjugate is at or about 20,000 Dto at or about 300,000 D in molecular weight, or is at or about 30,000 Dto at or about 300,000 D in molecular weight, or is at or about 40,000 Dto at or about 300,000 D in molecular weight, and wherein the conjugatecontains no more than about 10 PEG molecules, or no more than about 5PEG molecules, or no more than about 4 PEG molecules, or no more thanabout 3 PEG molecules, or no more than about 2 PEG molecules, or no morethan 1 PEG molecule.

In another aspect, the invention encompasses a conjugate containing anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH and F(ab′)₂, wherein the antibody fragment is derivatized withPEG, wherein every PEG molecule in the conjugate is at or about 20,000 Dto at or about 100,000 D in molecular weight, or is at or about 30,000 Dto at or about 100,000 D in molecular weight, or is at or about 40,000 Dto at or about 100,000 D in molecular weight, and wherein the conjugatecontains no more than about 10 PEG molecules, or no more than about 5PEG molecules, or no more than about 4 PEG molecules, or no more thanabout 3 PEG molecules, or no more than about 2 PEG molecules, or no morethan 1 PEG molecule.

In another aspect, the invention encompasses a conjugate containing anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH and F(ab′)₂, wherein the antibody fragment is derivatized withPEG, wherein every PEG molecule in the conjugate is at or about 20,000 Dto at or about 70,000 D in molecular weight, or is at or about 30,000 Dto at or about 70,000 D in molecular weight, or is at or about 40,000 Dto at or about 70,000 D in molecular weight, and wherein the conjugatecontains no more than about 10 PEG molecules, or no more than about 5PEG molecules, or no more than about 4 PEG molecules, or no more thanabout 3 PEG molecules, or no more than about 2 PEG molecules, or no morethan 1 PEG molecule.

In another aspect, the invention encompasses a conjugate containing anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH and F(ab′)₂, wherein the antibody fragment is derivatized withPEG, wherein every PEG molecule in the conjugate is at or about 20,000 Dto at or about 50,000 D in molecular weight, or is at or about 30,000 Dto at or about 50,000 D in molecular weight, or is at or about 40,000 Dto at or about 50,000 D in molecular weight, and wherein the conjugatecontains no more than about 10 PEG molecules, or no more than about 5PEG molecules, or no more than about 4 PEG molecules, or no more thanabout 3 PEG molecules, or no more than about 2 PEG molecules, or no morethan 1 PEG molecule.

In another aspect, the invention encompasses a conjugate containing anantibody fragment selected from the group consisting of Fab, Fab′,Fab′-SH and F(ab′)₂, wherein the antibody fragment is derivatized withPEG, wherein every PEG molecule in the conjugate is at or about 20,000 Dto at or about 40,000 D in molecular weight, or is at or about 30,000 Dto at or about 40,000 D in molecular weight, and wherein the conjugatecontains no more than about 10 PEG molecules, or no more than about 5PEG molecules, or no more than about 4 PEG molecules, or no more thanabout 3 PEG molecules, or no more than about 2 PEG molecules, or no morethan 1 PEG molecule.

In a preferred embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is derivatized with PEG having a molecular weightof at least about 20,000 D, or at least about 30,000 D, or at leastabout 40,000 D, and wherein every PEG molecule in the conjugate isattached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG having a molecularweight that is at or about 20,000 D to about 300,000 D, or is at orabout 30,000 D to at or about 300,000 D, or is at or about 40,000 D toat or about 300,000 D, and wherein every PEG molecule in the conjugateis attached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG having a molecularweight that is at or about 20,000 D to about 100,000 D, or is at orabout 30,000 D to at or about 100,000 D, or is at or about 40,000 D toat or about 100,000 D, and wherein every PEG molecule in the conjugateis attached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG having a molecularweight that is at or about 20,000 D to about 70,000 D, or is at or about30,000 D to at or about 70,000 D, or is at or about 40,000 D to at orabout 70,000 D, and wherein every PEG molecule in the conjugate isattached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG having a molecularweight that is at or about 20,000 D to about 50,000 D, or is at or about30,000 D to at or about 50,000 D, or is at or about 40,000 D to at orabout 50,000 D, and wherein every PEG molecule in the conjugate isattached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG having a molecularweight that is at or about 20,000 D to about 40,000 D, or is at or about30,000 D to at or about 40,000 D, and wherein every PEG molecule in theconjugate is attached to the hinge region of the antibody fragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at least about 20,000 D in molecularweight, or at least about 30,000 D in molecular weight, or at leastabout 40,000 D in molecular weight, wherein every PEG molecule in theconjugate molecule is attached to the hinge region of the antibodyfragment, and wherein the conjugate contains no more than about 10 PEGmolecules, or no more than about 5 PEG molecules, or no more than about4 PEG molecules, or no more than about 3 PEG molecules, or no more thanabout 2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 300,000D in molecular weight, or is at or about 30,000 D to at or about 300,000D in molecular weight, or is at or about 40,000 D to at or about 300,000D in molecular weight, wherein every PEG molecule in the conjugatemolecule is attached to the hinge region of the antibody fragment, andwherein the conjugate contains no more than about 10 PEG molecules, orno more than about 5 PEG molecules, or no more than about 4 PEGmolecules, or no more than about 3 PEG molecules, or no more than about2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 100,000D in molecular weight, or is at or about 30,000 D to at or about 100,000D in molecular weight, or is at or about 40,000 D to at or about 100,000D in molecular weight, wherein every PEG molecule in the conjugatemolecule is attached to the hinge region of the antibody fragment, andwherein the conjugate contains no more than about 10 PEG molecules, orno more than about 5 PEG molecules, or no more than about 4 PEGmolecules, or no more than about 3 PEG molecules, or no more than about2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 70,000D in molecular weight, or is at or about 30,000 D to at or about 70,000D in molecular weight, or is at or about 40,000 D to at or about 70,000D in molecular weight, wherein every PEG molecule in the conjugatemolecule is attached to the hinge region of the antibody fragment, andwherein the conjugate contains no more than about 10 PEG molecules, orno more than about 5 PEG molecules, or no more than about 4 PEGmolecules, or no more than about 3 PEG molecules, or no more than about2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 50,000D in molecular weight, or is at or about 30,000 D to at or about 50,000D in molecular weight, or is at or about 40,000 D to at or about 50,000D in molecular weight, wherein every PEG molecule in the conjugatemolecule is attached to the hinge region of the antibody fragment, andwherein the conjugate contains no more than about 10 PEG molecules, orno more than about 5 PEG molecules, or no more than about 4 PEGmolecules, or no more than about 3 PEG molecules, or no more than about2 PEG molecules, or no more than 1 PEG molecule.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 40,000D in molecular weight, or is at or about 30,000 D to at or about 40,000D in molecular weight, wherein every PEG molecule in the conjugatemolecule is attached to the hinge region of the antibody fragment, andwherein the conjugate contains no more than about 10 PEG molecules, orno more than about 5 PEG molecules, or no more than about 4 PEGmolecules, or no more than about 3 PEG molecules, or no more than about2 PEG molecules, or no more than 1 PEG molecule.

In yet another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at least about 20,000 D in molecular weight, or atleast about 30,000 D in molecular weight, or at least about 40,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 300,000 D inmolecular weight, or is at or about 30,000 D to at or about 300,000 D inmolecular weight, or is at or about 40,000 D to at or about 300,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 100,000 D inmolecular weight, or is at or about 30,000 D to at or about 100,000 D inmolecular weight, or is at or about 40,000 D to at or about 100,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 70,000 D inmolecular weight, or is at or about 30,000 D to at or about 70,000 D inmolecular weight, or is at or about 40,000 D to at or about 70,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 50,000 D inmolecular weight, or is at or about 30,000 D to at or about 50,000 D inmolecular weight, or is at or about 40,000 D to at or about 50,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains a F(ab′)₂antibody fragment derivatized with PEG, wherein every PEG molecule inthe conjugate is at or about 20,000 D to at or about 40,000 D inmolecular weight, or is at or about 30,000 D to at or about 40,000 D inmolecular weight, wherein the antibody fragment is attached to no morethan about 2 PEG molecules, and wherein every PEG molecule is attachedto a cysteine residue in the light or heavy chain of the antibodyfragment that would ordinarily form the disulfide bridge linking thelight and heavy chains, wherein the disulfide bridge is avoided bysubstituting another amino acid, such as serine, for the correspondingcysteine residue in the opposite chain.

In still another preferred embodiment, the conjugate contains anantibody fragment selected from the group consisting of Fab, Fab′, andFab′-SH, wherein the antibody fragment is derivatized with PEG, whereinevery PEG molecule in the conjugate is at least about 20,000 D inmolecular weight, or at least about 30,000 in molecular weight, or atleast about 40,000 D in molecular weight, wherein the antibody fragmentis attached to no more than 1 PEG molecule, and wherein the PEG moleculeis attached to a cysteine residue in the light or heavy chain of theantibody fragment that would ordinarily form the disulfide bridgelinking the light and heavy chains, wherein the disulfide bridge isavoided by substituting another amino acid, such as serine, for thecorresponding cysteine residue in the opposite chain.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 300,000D in molecular weight, or is at or about 30,000 D to at or about 300,000D in molecular weight, or is at or about 40,000 D to at or about 300,000D in molecular weight, wherein the antibody fragment is attached to nomore than 1 PEG molecule, and wherein the PEG molecule is attached to acysteine residue in the light or heavy chain of the antibody fragmentthat would ordinarily form the disulfide bridge linking the light andheavy chains, wherein the disulfide bridge is avoided by substitutinganother amino acid, such as serine, for the corresponding cysteineresidue in the opposite chain.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 100,000D in molecular weight, or is at or about 30,000 D to at or about 100,000D in molecular weight, or is at or about 40,000 D to at or about 100,000D in molecular weight, wherein the antibody fragment is attached to nomore than 1 PEG molecule, and wherein the PEG molecule is attached to acysteine residue in the light or heavy chain of the antibody fragmentthat would ordinarily form the disulfide bridge linking the light andheavy chains, wherein the disulfide bridge is avoided by substitutinganother amino acid, such as serine, for the corresponding cysteineresidue in the opposite chain.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 70,000D in molecular weight, or is at or about 30,000 D to at or about 70,000D in molecular weight, or is at or about 40,000 D to at or about 70,000D in molecular weight, wherein the antibody fragment is attached to nomore than 1 PEG molecule, and wherein the PEG molecule is attached to acysteine residue in the light or heavy chain of the antibody fragmentthat would ordinarily form the disulfide bridge linking the light andheavy chains, wherein the disulfide bridge is avoided by substitutinganother amino acid, such as serine, for the corresponding cysteineresidue in the opposite chain.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 50,000D in molecular weight, or is at or about 30,000 D to at or about 50,000D in molecular weight, or is at or about 40,000 D to at or about 50,000D in molecular weight, wherein the antibody fragment is attached to nomore than 1 PEG molecule, and wherein the PEG molecule is attached to acysteine residue in the light or heavy chain of the antibody fragmentthat would ordinarily form the disulfide bridge linking the light andheavy chains, wherein the disulfide bridge is avoided by substitutinganother amino acid, such as serine, for the corresponding cysteineresidue in the opposite chain.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is derivatized with PEG, wherein every PEGmolecule in the conjugate is at or about 20,000 D to at or about 40,000D in molecular weight, or is at or about 30,000 D to at or about 40,000D in molecular weight, wherein the antibody fragment is attached to nomore than 1 PEG molecule, and wherein the PEG molecule is attached to acysteine residue in the light or heavy chain of the antibody fragmentthat would ordinarily form the disulfide bridge linking the light andheavy chains, wherein the disulfide bridge is avoided by substitutinganother amino acid, such as serine, for the corresponding cysteineresidue in the opposite chain.

It will be appreciated that all of the above-described embodiments ofthe invention utilizing PEG polymers include conjugates wherein the PEGpolymer(s) is (are) linear or branched. In a preferred embodiment, theconjugate contains an antibody fragment selected from the groupconsisting of Fab, Fab′, and Fab′-SH, wherein the antibody fragment isattached to no more than 1 PEG molecule, and wherein the PEG molecule isbranched and at least about 40,000 D in molecular weight. In aparticularly surprising and unexpected finding, the inventors discoveredthat the foregoing conjugate exhibits a serum half-life, MRT and serumclearance rate approaching that of full length antibody as shown inExample X below.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 40,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 40,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 40,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 40,000 D to at or about 50,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and atleast 40,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 40,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 40,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 40,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 40,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In a preferred embodiment, the conjugate contains an antibody fragmentselected from the group consisting of Fab, Fab′, and Fab′-SH, whereinthe antibody fragment is attached to no more than 1 PEG molecule, andwherein the PEG molecule is linear and at least about 40,000 D inmolecular weight.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 40,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 40,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 40,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 40,000 D to at or about 50,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and atleast 40,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 40,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 40,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 40,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 40,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at least about 30,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 30,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 30,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 30,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 30,000 D to at or about 50,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 30,000 D to at or about 40,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and atleast 30,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 30,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 30,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 30,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 30,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 30,000 D to at or about 40,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at least about 30,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 30,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 30,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 30,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 30,000 D to at or about 50,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 30,000 D to at or about 40,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and atleast 30,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 30,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 30,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 30,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 30,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 30,000 D to at or about 40,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at least about 20,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 50,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 40,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is linear and has a molecularweight that is at or about 20,000 D to at or about 30,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and atleast 20,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 40,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is linear and has amolecular weight that is at or about 20,000 D to at or about 30,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at least about 20,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 300,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 100,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 70,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 50,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 40,000 D.

In another preferred embodiment, the conjugate contains an antibodyfragment selected from the group consisting of Fab, Fab′, and Fab′-SH,wherein the antibody fragment is attached to no more than 1 PEGmolecule, and wherein the PEG molecule is branched and has a molecularweight that is at or about 20,000 D to at or about 30,000 D.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and atleast 20,000 D in molecular weight, and the PEG molecule is attached tothe hinge region of the antibody fragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 300,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 100,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 70,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 50,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 40,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In another preferred embodiment, the invention provides a conjugatecontaining an antibody fragment selected from the group consisting ofFab, Fab′, and Fab′-SH, wherein the antibody fragment is attached to nomore than 1 PEG molecule, wherein the PEG molecule is branched and has amolecular weight that is at or about 20,000 D to at or about 30,000 D,and the PEG molecule is attached to the hinge region of the antibodyfragment.

In one aspect, the invention provides any of the above-describedconjugates wherein the conjugate contains no more than one antibodyfragment. Additionally provided herein is any of the above-describedconjugates wherein the conjugate contains one or more antibodyfragment(s) covalently linked to one or more polymer molecule(s), suchas conjugates containing two or more antibody fragments covalentlylinked together by polymer molecule(s). In one embodiment, a polymermolecule is used to link together two antibody fragments to form adumbbell-shaped structure. Also encompassed herein are conjugates formedby more than two antibody fragments joined by polymer molecule(s) toform a rosette or other shapes. The antibody fragments in suchstructures can be of the same or different fragment type and can havethe same antigen specificity or have different antigen specificities.Such structures can be made by using a polymer molecule derivatized withmultiple functional groups permitting the direct attachment, or theattachment by means of bi- or multi-functional linkers, of two or moreantibody fragments to the polymer backbone.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to rabbit IL-8 and/or human IL-8. In yetanother aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising 6G4.2.5LV/L1N35A or6G4.2.5LV/L1N35E as defined below. In still another aspect, theinvention encompasses any of the above-described conjugates utilizing anantibody fragment comprising 6G4.5.2.5HV11 as defined below. In afurther aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising hu6G4.2.5LV/L1N35Aor hu6G4.2.5LV/L1N35E as defined below. In an additional aspect, theinvention encompasses any of the above-described conjugates utilizing anantibody fragment comprising hu6G4.2.5HV. Further encompassed herein areany of the above-described conjugates utilizing an antibody fragmentcomprising 6G4.2.5LV/L1N35A or 6G4.2.5LV/L1N35E and further comprisingthe CDRs of 6G4.2.5HV as defined below. Also encompassed herein are anyof the above described conjugates utilizing an antibody fragmentcomprising hu6G4.2.5LV/L1N35A or hu6G4.2.5LV/L1N35E and furthercomprising hu6G4.2.5HV as defined below. Additionally encompassed hereinare any of the above-described conjugates utilizing an antibody fragmentcomprising 6G4.2.5LV11N35A or 6G4.2.5LV11N35E as defined below. Furtherprovided herein are any of the above-described conjugates utilizing anantibody fragment comprising 6G4.2.5LV11N35A or 6G4.2.5LV11N35E andfurther comprising 6G4.2.5HV11 as defined below.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human VEGF. In another embodiment, theforegoing antibody fragment competes with VEGF receptor for binding toVEGF. Such anti-VEGF antagonistic antibody fragments are used toconstruct conjugates that are capable of inhibiting one or more of thebiological activities of VEGF, for example, its mitogenic or angiogenicactivity. Antagonists of VEGF act by interfering with the binding ofVEGF to a cellular receptor, by incapacitating or killing cells whichhave been activated by VEGF, or by interfering with vascular endothelialcell activation after VEGF binding to a cellular receptor. All suchpoints of intervention used by anti-VEGF antagonists are also suitabletherapeutic targets for the anti-VEGF antibody fragment-polymerconjugates of the invention. Anti-human VEGF antibodies capable ofinterfering with the binding of VEGF to a cellular receptor aredescribed in WO 98/45331 published Oct. 15, 1998 (InternationalApplication No. PCT/US98/06604 filed Apr. 3, 1998).

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to HER2. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of thehuman ErbB2 (HER2) receptor. In yet another embodiment, the foregoingantibody fragment is capable of inducing cell death or apoptosis of aHER2-expressing cell. In still another embodiment, the foregoingconjugate utilizing an anti-HER2 antibody fragment further incorporatesa radioimaging or radiotherapeutic agent, including radionuclides suchas ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and ¹⁸⁶Re, or other nonproteinaceousdiagnostic label or chemotherapeutic agent, including small moleculetoxins such as calicheamicins, maytansinoids, palytoxins, trichothenes,and CC1065.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human CD20. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanCD20.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human CD18. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanCD18.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human CD11a. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanCD11a.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human IgE.

In another embodiment, the foregoing antibody fragment is capable ofcompeting with Fc_(∈)RI receptor for binding to human IgE, i.e. capableof inhibiting the binding of human IgE to the Fc_(∈)RI receptor. In yetanother embodiment, the foregoing antibody fragment binds tomembrane-bound IgE on the surface of human B-lymphocytes but does notbind to soluble IgE bound to Fc_(∈)RI receptor on the surface of humanbasophils.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human Apo-2 receptor. In anotherembodiment, the foregoing antibody fragment binds to the extracellulardomain of the human Apo-2 receptor. In yet another embodiment, theforegoing antibody fragment is capable of inducing cell death orapoptosis of an Apo-2 receptor-expressing cell.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human TNF-α.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human tissue factor.

In another apsect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human α₄β₇ integrin. In anotherembodiment, the foregoing antibody fragment binds to the extracellularregion of a human α₄β₇ integrin complex.

In another apsect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human GPIIb-IIIa integrin. In anotherembodiment, the foregoing antibody fragment binds to the extracellularregion of a human GPIIb-IIIa integrin complex.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human CD3. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanCD3.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human interleukin-2 receptor (IL-2R)α-chain (T-cell activation antigen or “TAC”). In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanTAC.

In another aspect, the invention encompasses any of the above-describedconjugates utilizing an antibody fragment comprising an antigenrecognition site that binds to human EGFR. In another embodiment, theforegoing antibody fragment binds to the extracellular domain of humanEGFR.

a. Production of Antibody Fragments

Antibody fragments can be produced by any method known in the art.Generally, an antibody fragment is derived from a parental intactantibody.

(i) Antigen Preparation

The antigen to be used for antibody generation can be prepared by anyconvenient method, such as recombinant methods. Membrane-bound proteinantigens can be presented by cell surface expression in recombinant ornon-recombinant cells, which cells can be used as immunogens for raisingthe desired antibody response against the membrane-bound proteinantigen. Alternatively, soluble forms of the membrane-bound proteinantigen can be generated, such as isolated extracellular domainfragments of membrane-anchored receptor proteins, or variants of suchreceptor proteins having deleted or inactivated transmembrane domains.In one embodiment, an extracellular domain is fused to the Fc region ofan immunoglobulin to form a chimeric protein immunogen.

A protein antigen of interest can be cloned, genetically engineered asdesired to add characteristics useful in antibody generation (such asfusion to an immunoglobulin Fc region), and produced in a recombinantexpression host cell system according to known methods. In oneembodiment, human VEGF-encoding DNA is obtained as described in U.S.Pat. No. 5,332,671 (issued Jul. 26, 1994) and used for production ofhuman VEGF in recombinant host cells according to the same generalmethods that are described for antibodies and antibody fragments inSection (II)(4) below, followed by recovery and purification of humanVEGF from recombinant host cell culture according to the same generalmethods that are described for antibodies and antibody fragments inSection (II)(4)(F) below. In another embodiment, human VEGF is obtainedas described in U.S. Pat. No. 5,332,671.

In one embodiment, soluble HER2-encoding DNA, such as HER2 extracellulardomain (ECD)-encoding DNA, is obtained as described in European PatentNo. 0 474 727 B1 (granted Jul. 23, 1997) (European regional phase of WO90/14357 published Nov. 29, 1990) and used for production of HER2 ECD inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (I)(4) below,followed by recovery and purification of human HER2 ECD from recombinanthost cell culture according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)(F)below. In another embodiment, HER2 ECD is obtained as described in EP 0474 727 B1.

In one embodiment, soluble human CD20-encoding DNA, such as human CD20extracellular domain (ECD)-encoding DNA, is obtained as described inTedder et al., “Isolation and Structure of a cDNA Encoding the B1 (CD20)Cell-Surface Antigen of Human B Lymphocytes,” Proc. Natl. Acad. Sci.(USA), 85: 208–212 (1988) and used for production of CD20 ECD inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (I)(4) below,followed by recovery and purification of human CD20 ECD from recombinanthost cell culture according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)(F)below.

In one embodiment, soluble human CD11a-encoding DNA, such as human CD11aI-domain-encoding DNA, is obtained as described in van Kooyk et al., J.Exp. Med., 183(3): 1247–1252 (1996), Edwards et al., J. Biol. Chem.,270(21): 12635–12640 (1995), or Champe et al., J. Biol. Chem., 270:1388–1394 (1995), and used for production of CD11a I-domain inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)below, followed by recovery and purification of human CD11a I-domainfrom recombinant host cell culture according to the same general methodsthat are described for antibodies and antibody fragments in Section(II)(4)(F) below.

In one embodiment, soluble human CD18-encoding DNA, such as human CD18extracellular domain (ECD)-encoding DNA, is obtained as described inKishimoto et al., “Cloning of the beta subunit of the leukocyte adhesionproteins: homology to an extracellular matrix receptor defines a novelsupergene family,” Cell, 48:681–690 (1987) and used for production ofCD18 ECD in recombinant host cells according to the same general methodsthat are described for antibodies and antibody fragments in Section(II)(4) below, followed by recovery and purification of human CD18 ECDfrom recombinant host cell culture according to the same general methodsthat are described for antibodies and antibody fragments in Section(II)(4)(F) below.

In one embodiment, human membrane-bound IgE extracellulardomain-encoding DNA is obtained as described in U.S. Pat. No. 5,091,131(issued Feb. 25, 1992) and used for production of human membrane-boundIgE ECD in recombinant host cells according to the same general methodsthat are described for antibodies and antibody fragments in Section(I)(4) below, followed by recovery and purification of humanmembrane-bound IgE ECD from recombinant host cell culture according tothe same general methods that are described for antibodies and antibodyfragments in Section (II)(4)(F) below. In another embodiment, humanmembrane-bound IgE ECD is obtained as described in U.S. Pat. No.5,091,131.

In one embodiment, soluble human Apo-2 receptor-encoding DNA, such ashuman Apo-2 receptor extracellular domain (ECD)-encoding DNA, isobtained as described in WO 98/51793 (published Nov. 19, 1998)(International Application No. PCT/US98/09704 filed May 14, 1998) andused for production of human Apo-2 receptor ECD in recombinant hostcells according to the same general methods that are described forantibodies and antibody fragments in Section (II)(4) below, followed byrecovery and purification of human Apo-2 receptor ECD from recombinanthost cell culture according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)(F)below.

In one embodiment, human TNF-α-encoding DNA is obtained as described inPennica et al., Nature, 512: 721 (1984) or U.S. Pat. No. 4,650,674(issued Mar. 17, 1987) and used for production of human TNF-α inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)below, followed by recovery and purification of human TNF-α fromrecombinant host cell culture according to the same general methods thatare described for antibodies and antibody fragments in Section(II)(4)(F) below. In another embodiment, human TNF-α is obtained asdescribed in U.S. Pat. No. 4,650,674.

In one embodiment, human tissue factor-encoding DNA is obtained asdescribed in European Patent No. 0 278 776 B1 (granted May 28, 1997) andused for production of human tissue factor in recombinant host cellsaccording to the same general methods that are described for antibodiesand antibody fragments in Section (II)(4) below, followed by recoveryand purification of human tissue factor from recombinant host cellculture according to the same general methods that are described forantibodies and antibody fragments in Section (II)(4)(F) below. Inanother embodiment, human tissue factor is obtained as described inEuropean Patent No. 0 278 776 B1.

In one embodiment, soluble human α₄ integrin-encoding DNA and solublehuman β₇ integrin encoding DNA, such as human α₄ integrin extracellulardomain (ECD)-encoding DNA along with human β₇ integrin extracellulardomain (ECD)-encoding DNA, are obtained as described in Takada et al.,“The primary structure of the α₄ subunit of VLA-4: Homology to otherintegrins and a possible cell-cell adhesion function”, EMBO J., 8:1361–1368 (1989) and Yuan et al., “Cloning and sequence analysis of anovel β₂-related integrin transcript from T lymphocytes: homology ofintegrin cysteine-rich repeates to domain III of laminin B chains”,International Immunology, 2: 1097–1108 (1990), respectively, and usedfor co-production of human α₄ integrin ECD and human β₇ integrin ECD inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)below, followed by recovery and purification of α₄ ECD-β₇ ECD complexfrom recombinant host cell culture according to the same general methodsthat are described for antibodies and antibody fragments in Section(II)(4)(F) below.

In one embodiment, soluble human GPIIb-encoding DNA and soluble humanGPIIIa-encoding DNA, such as human GPIIb extracellular domain(ECD)-encoding DNA along with human GPIIIa extracellular domain(ECD)-encoding DNA, are obtained as described in U.S. Pat. No. 5,726,037(issued Mar. 10, 1998) and used for co-production of human GPIIb ECD andhuman GPIIIa ECD in recombinant host cells according to the same generalmethods that are described for antibodies and antibody fragments inSection (II)(4) below, followed by recovery and purification of GPIIbECD-GPIIIa ECD complex from recombinant host cell culture according tothe same general methods that are described for antibodies and antibodyfragments in Section (II)(4)(F) below. Alternatively, human GPIIb-IIIacomplex can be produced and secreted from recombinant host cells asdescribed in Example 3 of U.S. Pat. No. 5,726,037.

In one embodiment, soluble human epidermal growth factor receptor(EGFR)-encoding DNA, such as human EGFR extracellular domain(ECD)-encoding DNA, is obtained as described in Ullrich et al., Nature,309: 418–425 (1984) and used for production of human EGFR ECD inrecombinant host cells according to the same general methods that aredescribed for antibodies and antibody fragments in Section (II)(4)below, followed by recovery and purification of human EGFR ECD fromrecombinant host cell culture according to the same general methods thatare described for antibodies and antibody fragments in Section(II)(4)(F) below.

In one embodiment, soluble human CD3-encoding DNA, such as human CD3extracellular domain (ECD)-encoding DNA, is obtained as described in vanden Elsen et al., “Isolation of cDNA clones encoding the 20K T3glycoprotein of human T-cell receptor complex,” Nature, 312:413–418(1984) and used for production of human CD3 ECD in recombinant hostcells according to the same general methods that are described forantibodies and antibody fragments in Section (II)(4) below, followed byrecovery and purification of human CD3 ECD from recombinant host cellculture according to the same general methods that are described forantibodies and antibody fragments in Section (II)(4)(F) below.

In one embodiment, soluble human interleukin-2 receptor (IL-2R) α-chain(T-cell activation antigen or “TAC”)-encoding DNA, such as human TACextracellular domain (ECD)-encoding DNA, is obtained as described inLeonard et al., Science, 230: 633–639 (1985) and used for production ofhuman TAC ECD in recombinant host cells according to the same generalmethods that are described for antibodies and antibody fragments inSection (II)(4) below, followed by recovery and purification of humanTAC ECD from recombinant host cell culture according to the same generalmethods that are described for antibodies and antibody fragments inSection (II)(4)(F) below.

(ii) Polyclonal Antibodies

The parental antibody can be generated by raising polyclonal seraagainst the desired antigen by multiple subcutaneous (sc) orintraperitoneal (ip) injections of antigen and an adjuvant, such asmonophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (RibiImmunochem. Research, Inc., Hamilton, Mont.), at multiple sites. Twoweeks later the animals are boosted. 7 to 14 days later animals are bledand the serum is assayed for anti-antigen titer. Animals are boosteduntil titer plateaus. Sera are harvested from animals, and polyclonalantibodies are isolated from sera by conventional immunoglobulinpurification procedures, such as protein A-Sepharose chromatography,hydroxylapatite chromatography, gel filtration, dialysis, or antigenaffinity chromatography. The desired antibody fragments can be generatedfrom purified polyclonal antibody preparations by conventional enzymaticmethods, e.g. F(ab′)₂ fragments are produced by pepsin cleavage ofintact antibody, and Fab fragments are produced by briefly digestingintact antibody with papain.

(iii) Monoclonal Antibodies

Alternatively, antibody fragments are derived from monoclonal antibodiesgenerated against the desired antigen. Monoclonal antibodies may be madeusing the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59–103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51–63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59–103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In one embodiment, anti-human VEGF monoclonal antibody is obtained asdescribed in WO 98/45331 (published Oct. 15, 1998) (InternationalApplication No. PCT/US98/06604 filed Apr. 3, 1998).

In another embodiment, anti-HER2 monoclonal antibody is obtained asdescribed in U.S. Pat. No. 5,725,856 (issued Mar. 10, 1998) orInternational Application No. CT/US98/26266 (filed Dec. 10, 1998).

In another embodiment, anti-human CD20 monoclonal antibody is obtainedas described in WO 94/11026 (published May 26, 1994) (InternationalApplication No. PCT/US93/10953 filed Nov. 12, 1993).

In another embodiment, anti-human CD18 monoclonal antibody is obtainedas described in U.S. Pat. No. 5,622,700 (issued Apr. 22, 1997). In yetanother embodiment, anti-human CD18 monoclonal antibody is obtained asdescribed in WO 97/26912 (published Jul. 31, 1997) (InternationalApplication No. PCT/US97/00492 filed Jan. 11, 1997).

In another embodiment, anti-human CD11a monoclonal antibody is obtainedas described in U.S. Pat. No. 5,622,700. In yet another embodiment,anti-human CD11a monoclonal antibody is obtained as described in WO98/23761 (published Jun. 4, 1998) (International Application No.PCT/US97/19041 filed Oct. 20, 1997).

In another embodiment, anti-human IgE monoclonal antibody is obtained asdescribed in U.S. Pat. No. 5,714,338 (issued Feb. 3, 1998). In yetanother embodiment, anti-human IgE monoclonal antibody is obtained asdescribed in U.S. Pat. No. 5,091,313 (issued Feb. 25, 1992). In stillanother embodiment, anti-human IgE monoclonal antibody is obtained asdescribed in WO 93/04173 (published Mar. 4, 1993) (InternationalApplication No. PCT/US92/06860 filed Aug. 14, 1992). In an additionalembodiment, anti-human IgE monoclonal antibody is obtained as describedin International Application No. PCT/US98/13410 (filed Jun. 30, 1998).In a further aspect, the invention comptemplates the use of anti-humanIgE monoclonal antibody capable of competing with Fc_(∈)RI receptor forbinding to human IgE, i.e. capable of inhibiting the binding of humanIgE to the Fc_(∈)RI receptor. Such anti-IgE monoclonal antibodies can beselected and identified by any convenient screening method, such anassay for inhibition of IgE-induced basophil cell sensitization asdescribed in U.S. Pat. No. 5,714,338.

In another embodiment, anti-human Apo-2 receptor monoclonal antibody isobtained as described in WO 98/51793 (published Nov. 19, 1998)(International Application No. PCT/US98/09704 filed May 14, 1998). In afurther embodiment, the invention contemplates the use of anti-humanApo-2 receptor monoclonal antibody capable of activating the human Apo-2receptor. Such anti-Apo-2 monoclonal antibodies can be selected andidentified by any convenient screening method, such as an assay forinduction of Apo-2 mediated 9D cell apoptosis as described in Example 10of WO 98/51793.

In another embodiment, anti-human TNF-α monoclonal antibody is obtainedas described in U.S. Pat. No. 5,672,347 (issued Sep. 30, 1997).

In another embodiment, anti-human tissue factor monoclonal antibody isobtained as described in European Patent No. 0 420 937 B1 (granted Nov.9, 1994).

In another embodiment, anti-human α₄-β₇ integrin monoclonal antibody isobtained as described in WO 98/06248 (published Feb. 19, 1998)(International Application No. PCT/US97/13884 filed Aug. 6, 1997).

In another embodiment, anti-human EGFR monoclonal antibody is obtainedas described in WO 96/40210 (published Dec. 19, 1996) (InternationalApplication No. PCT/US96/9847 filed Jun. 7, 1996).

In another embodiment, anti-human CD3 monoclonal antibody is obtained asdescribed in U.S. Pat. No. 4,515,893 (issued May 7, 1985).

In another embodiment, anti-human TAC monoclonal antibody is obtained asdescribed in U.S. Pat. No. 5,693,762 (issued Dec. 2, 1997).

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria ofantibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol.,5: 256 (1993) and Pluckthun, Immunol. Revs., 130: 151 (1992).

In a preferred embodiment, the antibody fragment is derived from ahumanized antibody. Methods for humanizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain. Itwill be appreciated that variable domain sequences obtained from anynon-human animal phage display library-derived Fv clone or from anynon-human animal hybridoma-derived antibody clone provided as describedherein can serve as the “import” variable domain used in theconstruction of the humanized antibodies of the invention. Humanizationcan be essentially performed following the method of Winter andco-workers (Jones et al., Nature, 321: 522 (1986); Riechmann et al.,Nature, 332: 323 (1988); Verhoeyen et al., Science, 239: 1534 (1988)),by substituting non-human animal, e.g. rodent, CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (Cabilly et al., supra),wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in non-human animal, e.g. rodent,antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a non-human animal, e.g. rodent, antibody isscreened against the entire library of known human variable-domainsequences. The human sequence which is closest to that of the non-humananimal is then accepted as the human framework (FR) for the humanizedantibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia and Lesk,J. Mol. Biol., 196: 901 (1987)). Another method uses a particularframework derived from the consensus sequence of all human antibodies ofa particular subgroup light or heavy chains. The same framework can beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci USA, 89: 4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)). It is also important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind to its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

In addition, antibody fragments for use herein can be derived from humanmonoclonal antibodies. Human monoclonal antibodies against the antigenof interest can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51–63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Alternatively, phage display technology (McCafferty et al., Nature348:552 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson et al., CurrentOpinion in Structural Biology 3:564 (1993). Several sources of V-genesegments can be used for phage display. Clackson et al., Nature 352:624(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581 (1991), orGriffith et al., EMBO J. 12:725 (1993). In a natural immune response,antibody genes accumulate mutations at a high rate (somatichypermutation). Some of the changes introduced will confer higheraffinity, and B cells displaying high-affinity surface immunoglobulinare preferentially replicated and differentiated during subsequentantigen challenge. This natural process can be mimicked by employing thetechnique known as “chain shuffling” (Marks et al., Bio/Technol. 10:779(1992)). In this method, the affinity of “primary” human antibodiesobtained by phage display can be improved by sequentially replacing theheavy and light chain V region genes with repertoires of naturallyoccurring variants (repertoires) of V domain genes obtained fromunimmunized donors. This technique allows the production of antibodiesand antibody fragments with affinities in the nM range. A strategy formaking very large phage antibody repertoires has been described byWaterhouse et al., Nucl. Acids Res. 21:2265 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

The invention also encompasses the use of bispecific and heteroconjugateantibody fragments having specificities for at least two differentantigens. Bispecific and heteroconjugate antibodies can be prepared asfull length antibodies or as antibody fragments (e.g. F(ab′)₂ bispecificantibody fragments). Antibody fragments having more than two valencies(e.g. trivalent or higher valency antibody fragments) are alsocontemplated for use herein. Bispecific antibodies, heteroconjugateantibodies, and multi-valent antibodies can be prepared as described inSection (II)(3)(C) below.

As described above, DNA encoding the monoclonal antibody or antibodyfragment of interest can be isolated from its hybridoma or phage displayclone of origin, and then manipulated to create humanized and/oraffinity matured constructs. In addition, known techniques can beemployed to introduce an amino acid residue or residues into any desiredlocation on the polypeptide backbone of the antibody fragment, e.g. acysteine residue placed in the hinge region of the heavy chain, therebyproviding a site for specific attachment of polymer molecule(s). In oneembodiment, the native cysteine residue in either the light or heavychain of the antibody fragment that would ordinarily form the disulfidebridge linking the light and heavy chains is substituted with anotheramino acid, such as serine, in order to leave the partner cysteineresidue in the opposite chain with a free suflhydryl for specificattachment of polymer molecule.

Upon construction of the desired antibody or antibody fragment-encodingclone, the clone can be used for recombinant production of the antibodyfragment as described in Section (II)(4) below. Finally, the antibody orantibody fragment product can be recovered from host cell culture andpurified as described in Section (II)(4)(F) below. In the case ofembodiments utilizing an antibody fragment engineered to lack a cysteineresidue that ordinarily forms the disulfide bridge between the light andheavy chains as described above, preferred recombinant productionsystems include bacterial expression and product recovery proceduresutilizing the low pH osmotic shock method described in the “AlternativeFab′-SH Purification” section of Example T below. If a full lengthantibody is produced, the desired antibody fragment can be obtainedtherefrom by subjecting the intact antibody to enzymatic digestionaccording to known methods, e.g. as described in Section (II)(4)(G)below.

b. Construction of Antibody Fragment-Polymer Conjugates

The antibody fragment-polymer conjugates of the invention can be made byderivatizing the desired antibody fragment with an inert polymer. Itwill be appreciated that any inert polymer which provides the conjugatewith the desired apparent size or which has the selected actual MW astaught herein is suitable for use in constructing the antibodyfragment-polymer conjugates of the invention.

Many inert polymers are suitable for use in pharmaceuticals. See, e.g.,Davis et al., Biomedical Polymers: Polymeric Materials andPharmaceuticals for Biomedical Use, pp. 441–451 (1980). In allembodiments of the invention, a non-proteinaceous polymer is used. Thenonproteinaceous polymer ordinarily is a hydrophilic synthetic polymer,i.e., a polymer not otherwise found in nature. However, polymers whichexist in nature and are produced by recombinant or in vitro methods arealso useful, as are polymers which are isolated from native sources.Hydrophilic polyvinyl polymers fall within the scope of this invention,e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly useful arepolyalkylene ethers such as polyethylene glycol (PEG); polyoxyalkylenessuch as polyoxyethylene, polyoxypropylene, and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides which comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble,but the final conjugate must be water soluble. Preferably, the conjugateexhibits a water solubility of at least about 0.01 mg/ml, and morepreferably at least about 0.1 mg/ml, and still more preferably at leastabout 1 mg/ml. In addition, the polymer should not be highly immunogenicin the conjugate form, nor should it possess viscosity that isincompatible with intravenous infusion or injection if the conjugate isintended to be administered by such routes.

In one embodiment, the polymer contains only a single group which isreactive. This helps to avoid cross-linking of protein molecules.However, it is within the scope herein to maximize reaction conditionsto reduce cross-linking, or to purify the reaction products through gelfiltration or ion exchange chromatography to recover substantiallyhomogenous derivatives. In other embodiments, the polymer contains twoor more reactive groups for the purpose of linking multiple antibodyfragments to the polymer backbone. Again, gel filtration or ion exchangechromatography can be used to recover the desired derivative insubstantially homogeneous form.

The molecular weight of the polymer can range up to about 500,000 D, andpreferably is at least about 20,000 D, or at least about 30,000 D, or atleast about 40,000 D. The molecular weight chosen can depend upon theeffective size of the conjugate to be achieved, the nature (e.g.structure, such as linear or branched) of the polymer, and the degree ofderivatization, i.e. the number of polymer molecules per antibodyfragment, and the polymer attachment site or sites on the antibodyfragment.

The polymer can be covalently linked to the antibody fragment through amultifunctional crosslinking agent which reacts with the polymer and oneor more amino acid residues of the antibody fragment to be linked.However, it is also within the scope of the invention to directlycrosslink the polymer by reacting a derivatized polymer with theantibody fragment, or vice versa.

The covalent crosslinking site on the antibody fragment includes theN-terminal amino group and epsilon amino groups found on lysineresidues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxylor other hydrophilic groups. The polymer may be covalently bondeddirectly to the antibody fragment without the use of a multifunctional(ordinarily bifunctional) crosslinking agent. Covalent binding to aminogroups is accomplished by known chemistries based upon cyanuricchloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxideplus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and aceticanhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde,activated succinimidyl esters, activated dithiocarbonate PEG,2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activatedPEG.) Carboxyl groups are derivatized by coupling PEG-amine usingcarbodiimide. Sulfhydryl groups are derivatized by coupling tomaleimido-substituted PEG (e.g. alkoxy-PEG amine plus sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) as described in WO97/10847 published Mar. 27, 1997, or PEG-maleimide commerciallyavailable from Shearwater Polymers, Inc., Huntsville, Ala.).Alternatively, free amino groups on the antibody fragment (e.g. epsilonamino groups on lysine residues) can be thiolated with 2-imino-thiolane(Traut's reagent) and then coupled to maleimide-containing derivativesof PEG as described in Pedley et al., Br. J. Cancer, 70: 1126–1130(1994).

The polymer will bear a group which is directly reactive with an aminoacid side chain, or the N- or C-terminus of the polypeptide linked, orwhich is reactive with the multifunctional cross-linking agent. Ingeneral, polymers bearing such reactive groups are known for thepreparation of immobilized proteins. In order to use such chemistrieshere, one should employ a water soluble polymer otherwise derivatized inthe same fashion as insoluble polymers heretofore employed for proteinimmobilization. Cyanogen bromide activation is a particularly usefulprocedure to employ in crosslinking polysaccharides.

“Water soluble” in reference to the starting polymer means that thepolymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.

The degree of substitution with such a polymer will vary depending uponthe number of reactive sites on the antibody fragment, the molecularweight, hydrophilicity and other characteristics of the polymer, and theparticular antibody fragment derivatization sites chosen. In general,the conjugate contains from 1 to about 10 polymer molecules, but greaternumbers of polymer molecules attached to the antibody fragments of theinvention are also contemplated. The desired amount of derivatization iseasily achieved by using an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the level of polymer substitution ofthe conjugates is determined by size exclusion chromatography or othermeans known in the art.

The polymer, e.g. PEG, is cross-linked to the antibody fragment by awide variety of methods known per se for the covalent modification ofproteins with nonproteinaceous polymers such as PEG. Certain of thesemethods, however, are not preferred for the purposes herein. Cyanuronicchloride chemistry leads to many side reactions, including proteincross-linking. In addition, it may be particularly likely to lead toinactivation of proteins containing sulfydryl groups. Carbonyldiimidazole chemistry (Beauchamp et al., Anal Biochem. 131, 25–33[1983]) requires high pH (>8.5), which can inactivate proteins.Moreover, since the “activated PEG” intermediate can react with water, avery large molar excess of “activated PEG” over protein is required. Thehigh concentrations of PEG required for the carbonyl diimidazolechemistry also led to problems in purification, as both gel filtrationchromatography and hydrophilic interaction chromatography are adverselyaffected. In addition, the high concentrations of “activated PEG” mayprecipitate protein, a problem that per se has been noted previously(Davis, U.S. Pat. No. 4,179,337). On the other hand, aldehyde chemistry(Royer, U.S. Pat. No. 4,002,531) is more efficient since it requiresonly a 40-fold molar excess of PEG and a 1–2 hr incubation. However, themanganese dioxide suggested by Royer for preparation of the PEG aldehydeis problematic “because of the pronounced tendency of PEG to formcomplexes with metal-based oxidizing agents” (Harris et al., J. Polym.Sci. Polym. Chem. Ed. 22, 341–52 [1984]). The use of a Moffattoxidation, utilizing DMSO and acetic anhydride, obviates this problem.In addition, the sodium borohydride suggested by Royer must be used athigh pH and has a significant tendency to reduce disulfide bonds. Incontrast, sodium cyanoborohydride, which is effective at neutral pH andhas very little tendency to reduce disulfide bonds is preferred. Inanother preferred embodiment, maleimido-activated PEG is used forcoupling to free thiols on the antibody fragment.

Functionalized PEG polymers to modify the antibody fragments of theinvention are available from Shearwater Polymers, Inc. (Huntsville,Ala.). Such commercially available PEG derivatives include, but are notlimited to, amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol,PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG aminoacids, PEG succinimidyl succinate, PEG succinimidyl propionate,succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate ofPEG, succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole,PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether,PEG-aldehyde, PEG vinylsulfone, PEG-maleimide,PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinylderivatives, PEG silanes, and PEG phospholides. The reaction conditionsfor coupling these PEG derivatives will vary depending on the protein,the desired degree of PEGylation, and the PEG derivative utilized. Somefactors involved in the choice of PEG derivatives include: the desiredpoint of attachment (such as lysine or cysteine R-groups), hydrolyticstability and reactivity of the derivatives, stability, toxicity andantigenicity of the linkage, suitability for analysis, etc. Specificinstructions for the use of any particular derivative are available fromthe manufacturer.

The conjugates of this invention are separated from the unreactedstarting materials by gel filtration or ion exchange HPLC. Heterologousspecies of the conjugates are purified from one another in the samefashion.

The conjugates may also be purified by ion-exchange chromatography. Thechemistry of many of the electrophilically activated PEG's results in areduction of amino group charge of the PEGylated product. Thus, highresolution ion exchange chromatography can be used to separate the freeand conjugated proteins, and to resolve species with different levels ofPEGylation. In fact, the resolution of different species (e.g.containing one or two PEG residues) is also possible due to thedifference in the ionic properties of the unreacted amino acids. In oneembodiment, species with difference levels of PEGylation are resolvedaccording to the methods described in WO 96/34015 (InternationalApplication No. PCT/US96/05550 published Oct. 31, 1996).

In a preferred embodiment, the conjugate is generated by utilizing thederivatization and purification methods described in Section (T) of theExamples below.

In one aspect, the invention provides any of the above-describedconjugates formed by its component parts, i.e. one or more antibodyfragment(s) covalently attached to one or more polymer molecule(s),without any extraneous matter in the covalent molecular structure of theconjugate.

c. Other Derivatives of Large Effective Size Conjugates

In another aspect, any of the above-described conjugates can be modifiedto contain one or more component(s) in addition to the antibody fragmentcomponent(s) and polymer component(s) that form the conjugate, whereinthe modification does not alter the essential functional property of theconjugate, namely, the substantially improved serum half-life, MRTand/or serum clearance rate as compared to that of the parental antibodyfragment from which the conjugate is derived. In one embodiment, theinvention provides any of the above-described conjugates modified toincorporate one or more nonproteinaceous functional group(s). Forexample, the conjugate can be modified to incorporate nonproteinaceouslabels or reporter molecules, such as radiolabels, including anyradioactive substance used in medical treatment or imaging or used as aneffector function or tracer in an animal model, such as radioisotopiclabels ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸ F,³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At,²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, ⁴⁰K, and the like, non-radioisotopic labelssuch as ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, ⁵⁶Fe, etc., fluroescent or chemiluminescentlabels, including fluorophores such as rare earth chelates, fluoresceinand its derivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels, stablefree radicals, and the like.

Conventional methods are available to bind these labels covalently tothe polypeptide antibody fragment or polymer component of the conjugate.In one aspect, any conjugate of the invention is modified byderivatizing the antibody fragment component with any of theabove-described non-proteinaceous labels, wherein the label is directlyor indirectly (through a coupling agent) attached to the antibodyfragment, and wherein such derivatization of the antibody fragment doesnot contribute or introduce any polymer moiety into the molecularstructure of the conjugate. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like can be used to tag the antibody fragment withthe above-described fluorescent or chemiluminescent labels. See, forexample, U.S. Pat. No. 3,940,475 (fluorimetry), Morrison, Meth.Enzymol., 32b, 103 (1974), Svyanen et al., J. Biol. Chem., 284, 3762(1973), and Bolton and Hunter, Biochem. J., 133, 529 (1973).

In the case of embodiments utilizing radiolabels, both direct andindirect labeling can be used to incorporate the selected radionuclideinto the conjugate. As used herein in the context of radiolabeling, thephrases “indirect labeling” and “indirect labeling approach” both meanthat a chelating agent is covalently attached to the antibody fragmentmoiety or polymer moiety of the conjugate and at least one raidonuclideis inserted into the chelating agent. Preferred chelating agents andradionuclides are set forth in Srivagtava, S. C. and Mease, R. C.,“Progress in Research on Ligands, Nuclides and Techniques for LabelingMonoclonal Antibodies,” Nucl. Med. Bio., 18(6): 589–603 (1991). Aparticularly preferred chelating agent is1-isothiocycmatobenzyl-3-methyldiothelene triaminepent acetic acid(“MX-DTPA”). As used herein in the context of radiolabeling, the phrases“direct labeling” and “direct labeling approach” both mean that aradionuclide is covalently attached directly to the antibody fragmentmoiety (typically via an amino acid residue) or to the polymer moiety ofthe conjugate. Preferred radionuclides for use in direct labeling ofconjugate are provided in Srivagtava and Mease, supra. In oneembodiment, the conjugate is directly labeled with ¹³¹I covalentlyattached to tyrosine residues. In another embodiment, the antibodyfragment component of the conjugate is directly or indirectly labeledwith any of the above-described radiolabels, wherein such labeling ofthe antibody fragment does not contribute or introduce any polymermoiety into the molecular structure of the conjugate.

In another embodiment, the conjugate can be modified to incorporate oneor more small molecule toxins, such as a calicheamicin, a maytansine(U.S. Pat. No. 5,208,020, expressly incorporated herein by reference),palytoxin, a trichothene, and CC1065. For example, the conjugate of theinvention can be derivatized with one or more maytansine molecules (e.g.about 1 to about 10 maytansine molecules per antibody molecule).Maytansine can be converted to May-ss-Me, which can be reduced toMay-SH3 and reacted with modified antibody fragment to generate amaytansinoid-derivatized antibody fragment moiety in the conjugate.

In yet another embodiment, the antibody fragment in the conjugate isderivatized with one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structural analogues ofcalicheamicin which may be used include, but are not limited to, ₁ ^(l),₂ ^(l), ₃ ^(l), N-acetyl-₁ ^(l), PSAG and ^(l) ₁ (Hinman et al., CancerR. 53:3336–3342 [1993]; and Lode et al., Cancer R. 58:2925–2928 [1998]).

d. Therapeutic Compositions and Administration of Large Effective SizeConjugates

The conjugate of the invention is useful for treating the diseaseindications that are treated with the parent intact antibody. In oneaspect, the invention provides the use of conjugates derived from aparental antibody that binds to an effector molecule selected from thegroup consisint of human VEGF, HER2, human CD20, human CD18, humanCD11a, human IgE, human Apo-2 receptor, human TNF-α, human tissuefactor, human α₄β₇ integrin, human GPIIb-IIIa integrin, human EGFR,human CD3, human IL-2R α-chain, and human IL-8 in the treatment of adisease that is mediated by the effector molecule.

(i) VEGF-Mediated Disorders

In one embodiment, the invention provides a method for treating aVEGF-mediated disease in a human patient with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human VEGF. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of VEGF-mediated disorders,including pathologies supported by blood vessel proliferation, i.e.angiogenesis, in a manner similar to the application of anti-VEGFantibodies in the treatment of such disease indications that is known inthe art, which treatment indications include solid tumors ((Kim et al.Nature 362:841–844 (1993); Warren et al. J. Clin. Invest. 95:1789–1797(1995); Borgström et al. Cancer Res. 56:4032–4039 (1996); and Melnyk etal. Cancer Res. 56:921–924 (1996)) and intraocular neovascular syndromessuch as proliferative retinopathies and age-related macular degeneration(AMD) (Adamis et al. Arch. Ophthalmol. 114:66–71 (1996)).

As shown in the Examples below, the conjugates of the inventionapproximate the in vivo pharmacokinetics (e.g. serum half-life,clearance and mean residence time as shown in FIGS. 72–73 and in ExampleAB below) and the in vivo therapeutic efficacy (e.g. the treatment ofsolid tumors as shown in FIG. 74 and in Example AC below) of full lengthanti-VEGF monoclonal antibody. Since conjugates of the invention derivedfrom anti-VEGF antibodies and fragments display the same orsubstantially similar in vivo activities as full length anti-VEGFmonoclonal antibody across a range of different parameters, includingpharmacokinetic characteristics and therapeutic endpoints in an animaltumor model, the data support the efficacy of the conjugates in the samebroad spectrum of neovascular disease indications that responds to fulllength anti-VEGF antibody treatment.

As noted above, any conjugate described in this Section (II) that isderived from an anti-VEGF antibody or fragment can be advantageouslyutilized in a method of treating a VEGF-mediated disease or disorder,such as neovascular disorders. In one embodiment, the invention providesa method of treating a neovascular disorder in a human patientcomprising administering to the patient an effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human VEGF.

In another embodiment, the invention provides a method of treating asolid tumor disorder in a human patient comprising administering to thepatient an effective amount of any conjugate described in this Section(II) wherein at least one antibody fragment in the conjugate comprisesan antigen binding site that binds to human VEGF. In yet anotherembodiment, the solid tumor disorder in the foregoing method is selectedfrom the group consisting of breast carcinomas, lung carcinomas, gastriccarcinomas, esophageal carcinomas, colorectal carcinomas, livercarcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervicalcarcinomas, endometrial carcinoma, endometrial hyperplasia,endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer,nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi'ssarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), and Meigs' syndrome.

In still another embodiment, the invention provides a method of treatingan intraocular neovascular disorder in a human patient comprisingadministering to the patient an effective amount of any conjugatedescribed in this Section (II) wherein at least one antibody fragment inthe conjugate comprises an antigen binding site that binds to humanVEGF. In a further embodiment, the intraocular neovascular disorder isselected from the group consisting of diabetic and other proliferativeretinopathies including retinopathy of prematurity, retrolentalfibroplasia, neovascular glaucoma, and age-related macular degeneration.

In another embodiment, the invention provides a method of inhibitingangiogenesis in a human patient comprising administering to the patientan effective amount of any conjugate described in this Section (II)wherein at least one antibody fragment in the conjugate comprises anantigen binding site that binds to human VEGF.

(ii) Disorders Mediated by HER2-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by HER2-expressing cells with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to HER2. Such conjugates have prophylactic and therapeuticapplications in a broad spectrum of HER2-expressing cell-mediateddisorders, including pathologies supported by the proliferation of cellsexpressing HER2, such as cancers characterized by overexpression ofHER2, in a manner similar to the application of full length anti-HER2antibodies in the treatment of such disease indications that is known inthe art, which treatment indications include HER2-overexpressing breast,ovarian and lung cancers.

In one embodiment, the invention provides a method of treating aHER2-expressing cell mediated disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto HER2. The disorder can be a HER2-expressing cell proliferativedisorder, including a benign or malignant tumor characterized by theoverexpression of the ErbB2 receptor, e.g. a cancer, such as, breastcancer, squamous cell cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,colon cancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer. In addition, the invention contemplates the use of the foregoingconjugate in place of full length anti-HER2 antibody in the treatment ofHER2-overexpressing cancers as described in U.S. Pat. No. 5,725,856 orInternational Patent Application No. PCT/US98/26266.

(iii) Disorders Mediated by CD20-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by CD20-expressing cells with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human CD20. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of CD20-expressingcell-mediated disorders, including pathologies supported by theproliferation of CD20-expressing cells, such as cancers ofCD20-expressing cells, in a manner similar to the application of fulllength anti-CD20 antibodies in the treatment of such disease indicationsthat is known in the art, which treatment indications includeB-lymphocytic lymphomas.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a CD20-expressing cell,comprising administering to the patient a therapeutically effectiveamount of any conjugate described in this Section (II) wherein at leastone antibody fragment in the conjugate comprises an antigen binding sitethat binds to human CD20. In another embodiment, the CD20-expressingcell-mediated disorder is a B-lymphocyte proliferative disorder, such asB-lymphocytic lymphoma. In addition, the invention contemplates the useof the foregoing conjugate in place of full length anti-CD20 antibody inthe treatment of B-lymphocyte proliferative disorders as described in WO94/11026 (published May 26, 1994) (International Application No.PCT/US93/10953 filed Nov. 12, 1993).

(iv) Disorders Mediated by CD18-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by CD18-expressing cells with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human CD18. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of CD18-expressingcell-mediated disorders, including pathologies supported by leukocyteadhesion, in a manner similar to the application of full lengthanti-CD18 antibodies in the treatment of such disease indications thatis known in the art, which treatment indications include acutemyocardial infarction and stroke.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a CD18-expressing cell,comprising administering to the patient a therapeutically effectiveamount of any conjugate described in this Section (II) wherein at leastone antibody fragment in the conjugate comprises an antigen binding sitethat binds to human CD18. In another embodiment, the CD18-expressingcell-mediated disorder is an inflammatory disorder, such as an ischemicreperfusion disorder, including acute myocardial infarction and stroke.In addition, the invention contemplates the use of the foregoingconjugate in place of full length anti-CD18 antibody in the treatment ofstroke as described in WO 97/26912.

In another embodiment, the invention provides a method of treating aLFA-1-mediated disorder in a human, comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human CD18. Inaddition, the invention contemplates the use of the foregoing conjgatein place of full length anti-CD18 antibody in the treatment of anLFA-1-mediated disorder, such as psoriasis and graft rejection, in ahuman patient as described in U.S. Pat. No. 5,622,700.

(v) Disorders Mediated by CD11a-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by a CD11a-expressing cell with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human CD11a. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of CD11a-expressingcell-mediated disorders, including pathologies supported by leukocyteadhesion, in a manner similar to the application of full lengthanti-CD11a antibodies in the treatment of such disease indications thatis known in the art, which treatment indications include psoriasis,asthma, graft rejection, and multiple sclerosis.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a CD11a-expressing cell,comprising administering to the patient a therapeutically effectiveamount of any conjugate described in this Section (II) wherein at leastone antibody fragment in the conjugate comprises an antigen binding sitethat binds to human CD11a.

In another embodiment, the invention provides a method of treating aninflammatory disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human CD11a.In another embodiment, the inflammatory disorder is psoriasis.

In another embodiment, the invention provides a method of treating animmune disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human CD11a.In still another embodiment, the immune disorder is graft rejection. Ina further embodiment, the immune disorder is multiple sclerosis.

In another embodiment, the invention provides a method of treatingasthma in a human patient comprising administering to the patient antherapeutically effective amount of any conjugate described in thisSection (II) wherein at least one antibody fragment in the conjugatecomprises an antigen binding site that binds to human CD11a.

In another embodiment, the invention provides a method of treating aLFA-1-mediated disorder in a human, comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human CD11a.In addition, the invention contemplates the use of the foregoingconjugate in place of full length anti-CD11a antibody in the treatmentof an LFA-1-mediated disorder, such as psoriasis and graft rejection, ina human patient as described in U.S. Pat. No. 5,622,700. In anotheraspect, the invention contemplates the use of the foregoing conjugate inplace of full length anti-CD11a antibody in the treatment ofLFA-1-mediated disorders in a human patient as described in WO 98/23761.

(vi) IgE-Mediated Disorders

In one embodiment, the invention provides a method for treating anIgE-mediated disorder in a human patient with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human IgE. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of IgE-mediated disorders,including pathologies characterized by the overproduction and/orhypersensitivity to the immunoglobulin IgE, in a manner similar to theapplication of anti-IgE antibodies in the treatment of such diseaseindications that is known in the art, which treatment indicationsinclude allergic diseases, such as allergic asthma and allergicrhinitis.

In one embodiment, the invention provides a method of treating anIgE-mediated disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human IgE. Inanother embodiment, the IgE-mediated disorder is an allergic disease. Inyet another embodiment, the IgE-mediated disorder is allergic asthma. Instill another embodiment, the IgE-mediated disorder is allergicrhinitis.

In a further embodiment, the invention provides a method of treating anIgE-mediated disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that competes with humanFc_(∈)RI for binding to human IgE. In yet another embodiment, theinvention provides a method of treating an IgE-mediated disorder in ahuman patient comprising administering to the patient a therapeuticallyeffective amount of any conjugate described in this Section (II) whereinat least one antibody fragment in the conjugate comprises an antigenbinding site that binds to membrane-bound IgE on the surface of humanB-lymphocytes but does not bind to soluble IgE bound to Fc_(∈)RIreceptor on the surface of human basophils. In addition, the inventioncontemplates the use of any of the foregoing conjugates in place of fulllength anti-human IgE antibody in the treatment of an IgE-mediateddisorder, such as allergic diseases including allergic asthma andallergic rhinitis, in a human patient as described in InternationalApplication No. PCT/US98/13410 (filed Jun. 30, 1998). In another aspect,the invention contemplates the use of any of the foregoing conjugates inplace of full length anti-human IgE antibody in the treatment ofallergic asthma in a human patient as described in WO 97/04807(published Feb. 13, 1997) (International Application No. PCT/US96/12275filed Jul. 24, 1996).

In another embodiment, the invention provides a method of treating anallergic disease in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that competes with humanFc_(∈)RI for binding to human IgE. In yet another embodiment, theinvention provides a method of treating an allergic disease in a humanpatient comprising administering to the patient a therapeuticallyeffective amount of any conjugate described in this Section (II) whereinat least one antibody fragment in the conjugate comprises an antigenbinding site that binds to membrane-bound IgE on the surface of humanB-lymphocytes but does not bind to soluble IgE bound to Fc_(∈)RIreceptor on the surface of human basophils.

In another embodiment, the invention provides a method of treatingallergic asthma in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that competes with humanFc_(∈)RI for binding to human IgE. In yet another embodiment, theinvention provides a method of treating allergic asthma in a humanpatient comprising administering to the patient a therapeuticallyeffective amount of any conjugate described in this Section (II) whereinat least one antibody fragment in the conjugate comprises an antigenbinding site that binds to membrane-bound IgE on the surface of humanB-lymphocytes but does not bind to soluble IgE bound to Fc_(∈)RIreceptor on the surface of human basophils.

In another embodiment, the invention provides a method of treatingallergic rhinitis in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that competes with humanFc_(∈)RI for binding to human IgE. In yet another embodiment, theinvention provides a method of treating allergic rhinitis in a humanpatient comprising administering to the patient a therapeuticallyeffective amount of any conjugate described in this Section (II) whereinat least one antibody fragment in the conjugate comprises an antigenbinding site that binds to membrane-bound IgE on the surface of humanB-lymphocytes but does not bind to soluble IgE bound to Fc_(∈)RIreceptor on the surface of human basophils.

(vii) Disorders Mediated by Cells Expressing Apo-2 Receptor

In one embodiment, the invention provides a method for treating a humandisease mediated by cells expressing Apo-2 receptor with any of theconjugates described in this Section (II) that is derived from aparental antibody that binds to human Apo-2 receptor. Such conjugateshave prophylactic and therapeutic applications in a broad spectrum ofApo-2 receptor-expressing cell-mediated disorders, including cancerssusceptible to Apo-2 receptor-mediated apoptosis, in a manner similar tothe application of full length anti-Apo-2 receptor antibodies in thetreatment of such disease indications that is known in the art, whichtreatment indications include cancers.

In one embodiment, the invention provides a method of treating aproliferative disorder in a human patient comprising administering tothe patient a therapeutically effective amount of any conjugatedescribed in this Section (II) wherein at least one antibody fragment inthe conjugate comprises an antigen binding site that binds to humanApo-2 receptor. The proliferative disorder can be a benign or malignanttumor characterized by cells expressing the Apo-2 receptor, e.g. acancer, such as breast cancer, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer.

In another embodiment, the invention provides a method of treating aproliferative disorder in a human patient comprising administering tothe patient a therapeutically effective amount of any conjugatedescribed in this Section (II) wherein at least one antibody fragment inthe conjugate comprises an antigen binding site that binds to the humanApo-2 receptor, and wherein the conjugate is an agonist of the humanApo-2 receptor, i.e. capable of inducing Apo-2 receptor-mediated cellapoptosis. The proliferative disorder can be a benign or malignant tumorcharacterized by cells expressing the Apo-2 receptor, e.g. a cancer,such as breast cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer. In addition, the inventioncontemplates the use of the foregoing conjugate in place of full lengthanti-Apo-2 receptor agonist antibody in the treatment of cancers, e.g.colon cancer, as described in WO 98/51793 (published Nov. 19, 1998)(International Application No. PCT/US98/09704 filed May 14, 1998).

(vi) TNF-α-Mediated Disorders

In one embodiment, the invention provides a method for treating aTNF-α-mediated disease with any of the conjugates described in thisSection (II) that is derived from a parental antibody that binds tohuman TNF-α. Such conjugates have prophylactic and therapeuticapplications in a broad spectrum of TNF-α-mediated disorders, includinginflammatory disorders and immune disorders, in a manner similar to theapplication of full length anti-human TNF-α antibodies in the treatmentof such disease indications that is known in the art, which treatmentindications include Crohn's disease, inflammatory bowel disease, andrheumatoid arthritis.

In one embodiment, the invention provides a method of treating aTNF-α-mediated disorder in a human patient comprising administering tothe patient a therapeutically effective amount of any conjugatedescribed in this Section (II) wherein at least one antibody fragment inthe conjugate comprises an antigen binding site that binds to humanTNF-α.

In one embodiment, the invention provides a method of treating aninflammatory disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human TNF-α.In another embodiment, the inflammatory disorder is Crohn's disease. Inyet another embodiment, the inflammatory disorder is inflammatory boweldisease. In still another embodiment, the inflammatory disorder isrheumatoid arthritis. In addition, the invention contemplates the use ofthe foregoing conjugate in place of full length anti-human TNF-αantibody in the treatment of TNF-α-mediated disorders, includinginflammatory disorders and immune disorders such as graft-versus-hostdisease (GHVD) as described in U.S. Pat. No. 5,672,347 (issued Sep. 30,1997). In another aspect, the invention contemplates the use of theforegoing conjugate in place of full length anti-human TNF-α antibody inthe treatment of Crohn's disease as described in U.S. Pat. No. 5,656,272(issued Aug. 12, 1997). In yet another aspect, the inventioncontemplates the use of the foregoing conjugate in place of full lengthanti-human TNF-α antibody in the treatment of rheumatoid arthritis asdescribed in U.S. Pat. No. 5,698,195 (issued Dec. 16, 1997).

(vii) Tissue Factor-Mediated Disorders

In one embodiment, the invention provides a method for treating a tissuefactor-mediated disease with any of the conjugates described in thisSection (II) that is derived from a parental antibody that binds tohuman tissue factor. Such conjugates have prophylactic and therapeuticapplications in a broad spectrum of tissue factor-mediated disorders,including pathologies supported by blood coagulation, in a mannersimilar to the application of full length anti-human tissue factorantibodies in the treatment of such disease indications that is known inthe art, which treatment indications include deep vein thrombosis andarterial thrombosis.

In one embodiment, the invention provides a method of treating a tissuefactor-mediated disorder in a human patient comprising administering tothe patient a therapeutically effective amount of any conjugatedescribed in this Section (II) wherein at least one antibody fragment inthe conjugate comprises an antigen binding site that binds to humantissue factor.

In one embodiment, the invention provides a method of treating athrombotic or prothrombotic disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human tissue factor. The thrombotic or prothrombotic disorder can beany disorder characteristically associated with a hyperthrombotic stateleading to intravascular thrombi or emboli, including diseases involvingvascular narrowing or occlusion, such as deep vein thrombosis, arterialthrombosis, atherosclerosis, vascular stenosis, myocardial ischemicdiseases including acute myocardial infarction, reocclusion followingangioplasty or atherectomy or thrombolytic treatment for acutemyocardial infarction, angina, cerebral ischemic diseases includingstroke, venous thrombophlebitis, and pulmonary embolism. In yet anotheraspect, the invention contemplates the use of the foregoing conjugate inplace of full length anti-human tissue factor antibody in the treatmentof thrombotic and prothrombotic diseases, such as coronary arterythrombotic diseases as described in European Patent No 0 420 937 B1(granted Nov. 19, 1994).

In another embodiment, the invention provides a method of inhibitingblood coagulation in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human tissuefactor.

(viii) Disorders Mediated by α4β7 Integrin-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by an α₄β₇ integrin-expressing cell with any of theconjugates described in this Section (II) that is derived from aparental antibody that binds to human α₄β₇ integrin. Such conjugateshave prophylactic and therapeutic applications in a broad spectrum ofα₄β₇ integrin-expressing cell-mediated disorders, including pathologiessupported by leukocyte adhesion, in a manner similar to the applicationof full length anti-α₄β₇ integrin antibodies in the treatment of suchdisease indications that is known in the art, which treatmentindications include inflammatory bowel disease.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by an α₄β₇ integrin-expressingcell, comprising administering to the patient a therapeuticallyeffective amount of any conjugate described in this Section (II) whereinat least one antibody fragment in the conjugate comprises an antigenbinding site that binds to human α₄β₇ integrin.

In another embodiment, the invention provides a method of treating aninflammatory disorder in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to human α₄β₇integrin. In another embodiment, the inflammatory disorder isinflammatory bowel disease. In another aspect, the inventioncontemplates the use of the foregoing conjugate in place of full lengthanti-human α₄β₇ integrin antibody in the treatment of inflammatorydisorders in a human patient as described in WO 98/06248 (published Feb.19, 1998) (International Patent Application No. PCT/US97/13884 filedAug. 6, 1997).

(ix) GPIIb-IIIa Integrin-Expressing Cell-Mediated Disorders

In one embodiment, the invention provides a method for treating a humandisease mediated by a GPIIb-IIIa integrin-expressing cell with any ofthe conjugates described in this Section (II) that is derived from aparental antibody that binds to human GPIIb-IIIa integrin. Suchconjugates have prophylactic and therapeutic applications in a broadspectrum of GPIIIa-IIIB integrin-expressing cell-mediated disorders,including pathologies supported by platelet aggregation, such asthrombotic disorders and prothrombotic disorders, in a manner similar tothe application of full length anti-human GPIIb-IIIa integrin antibodiesin the treatment of such disease indications that is known in the art,which treatment indications include unstable angina and reocclusionfollowing angioplasty or thrombolytic treatment of acute myocardialinfarction.

In one embodiment, the invention provides a method of treating athrombotic or prothrombotic disorder in a human patient comprisingadministering to the patient a therapeutically effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human GPIIb-IIIa integrin. The thrombotic or prothrombotic disordercan be any disorder characteristically associated with a hyperthromboticstate leading to intravascular thrombi or emboli, including diseasesinvolving vascular narrowing or occlusion, such as deep vein thrombosis,arterial thrombosis, atherosclerosis, vascular stenosis, myocardialischemic diseases including acute myocardial infarction, reocclusionfollowing angioplasty or atherectomy or thrombolytic treatment for acutemyocardial infarction, angina, cerebral ischemic diseases includingstroke, venous thrombophlebitis, and pulmonary embolism. In anotheraspect, the invention contemplates the use of the foregoing conjugate inplace of full-length anti-human GPIIb-IIIa antibody in a method forinhibition of thrombus formation in a human patient as described in U.S.Pat. No. 5,387,413 (issued Feb. 7, 1995). In yet another aspect, theinvention contemplates the use of the foregoing conjugate in place ofunpegylated anti-human GPIIb-IIIa antibody fragment, e.g. Fab, Fab′ orF(ab′)₂, in the treatment of thrombotic and prothrombotic diseases,including coronary artery thrombotic diseases such as restenosisfollowing percutaneous coronary artery transluminal angioplasty oratherectomy as described for REOPRO® abciximab in Physician's DeskReference, 52^(nd) Edition (1998), pp. 1498–1501.

In another embodiment, the invention provides a method of inhibitingblood coagulation in a human patient comprising administering to thepatient a therapeutically effective amount of any conjugate described inthis Section (II) wherein at least one antibody fragment in theconjugate comprises an antigen binding site that binds to humanGPIIb-IIIa integrin.

In still another embodiment, the invention provides a method ofinhibiting platelet aggregation in a human patient comprisingadministering to the patient a therapeutically effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human GPIIb-IIIa integrin.

(x) Disorders Mediated by EGFR-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by EGFR-expressing cells with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human EGFR. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of EGFR-expressingcell-mediated disorders, including pathologies supported by theproliferation of cells expressing EGFR, such as cancers characterized byoverexpression of EGFR, in a manner similar to the application of fulllength anti-EGFR antibodies in the treatment of such disease indicationsthat is known in the art, which treatment indications includeEGFR-overexpressing cancers of the breast, ovary, head and neck, brain,bladder, pancreas, and lung.

In one embodiment, the invention provides a method of treating a cellproliferation disorder in a human patient characterized byoverexpression of epidermal growth factor receptor (EGFR) comprisingadministering to the patient a therapeutically effective amount of anyconjugate described in this Section (II) wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human EGFR. The disorder can be a benign or malignant tumorcharacterized by the overexpression of the EGFR, e.g. a cancer, such as,breast cancer, squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer. In addition, the inventioncontemplates the use of the foregoing conjugate in place of full lengthanti-EGFR antibody in the treatment of EGFR-overexpressing cancers asdescribed in WO 96/40210 (published Dec. 19, 1996) (InternationalApplication No. PCT/US96/9847 filed Jun. 7, 1996).

(xi) Disorders Mediated by CD3-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by CD3-expressing cells with any of the conjugatesdescribed in this Section (II) that is derived from a parental antibodythat binds to human CD3. Such conjugates have prophylactic andtherapeutic applications in a broad spectrum of CD3-expressingcell-mediated disorders, including pathologies supported by theproliferation or activation of cells expressing CD3, such as immunedisorders mediated by T-lymphocytes, in a manner similar to theapplication of full length anti-human CD3 antibodies in the treatment ofsuch disease indications that is known in the art, which treatmentindications include graft rejection in transplant recipients.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a CD3-expressing cell,comprising administering to the patient a therapeutically effectiveamount of any conjugate described in this Section (II) wherein at leastone antibody fragment in the conjugate comprises an antigen binding sitethat binds to human CD3. In another embodiment, the CD3-expressingcell-mediated disorder is characterized by the activation orproliferation of T-lymphocytes, including immune disorders such as graftrejection in transplant recipients. In addition, the inventioncontemplates the use of the foregoing conjugate in place of full lengthanti-human CD3 antibody in the treatment of T-lymphocyte mediateddisorders as described in U.S. Pat. No. 4,515,893 (issued May 7, 1985).In another aspect, the invention contemplates the use of the foregoingconjugate in place of full length anti-human CD3 antibody in thetreatment of acute allograft rejection in renal transplant recipients asdescribed for ORTHOCLONE OKT3® muromonab-CD3 in Physician's DeskReference, 52^(nd) Edition (1998), pp. 1971–1974.

(xii) Disorders Mediated by TAC-Expressing Cells

In one embodiment, the invention provides a method for treating a humandisease mediated by interleukin-2 receptor α-chain (TAC)-expressingcells with any of the conjugates described in this Section (II) that isderived from a parental antibody that binds to human TAC. Suchconjugates have prophylactic and therapeutic applications in a broadspectrum of TAC-expressing cell-mediated disorders, includingpathologies supported by the proliferation or activation of cellsexpressing TAC, such as immune disorders mediated by T-lymphocytes orB-lymphocytes, in a manner similar to the application of full lengthanti-human TAC antibodies in the treatment of such disease indicationsthat is known in the art, which treatment indications include graftrejection in transplant recipients.

In one embodiment, the invention provides a method of treating adisorder in a human patient mediated by a TAC-expressing cell,comprising administering to the patient a therapeutically effectiveamount of any conjugate described in this Section (II) wherein at leastone antibody fragment in the conjugate comprises an antigen binding sitethat binds to human TAC. In another embodiment, the TAC-expressingcell-mediated disorder is characterized by the activation orproliferation of T-lymphocytes or B-lymphocytes, including immunedisorders such as graft rejection in transplant recipients. In addition,the invention contemplates the use of the foregoing conjugate in placeof full length anti-human TAC antibody in the treatment of T-lymphocyteor B-lymphocyte mediated disorders, including graft-versus-host disease(GHVD), graft rejection in transplant recipients, such as acute graftrejection in renal transplant recipients, and autoimmune diseases suchas Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemiclupus erythematosus, and myasthenia gravis, as described in U.S. Pat.No. 5,693,761.

(xiii) IL-8-Mediated Disorders

In one embodiment, the invention provides a method for treating anIL-8-mediated disease with any of the conjugates described in thisSection (II) that is derived from a parental antibody that binds torabbit or human IL-8. For example, a conjugate derived from an anti-IL-8antibody or fragment is useful in the treatment of inflammatorydisorders as described in Section (II)(5)(B) below. Such conjugates haveprophylactic and therapeutic applications in a broad spectrum of IL-8mediated diseases, such as inflammatory diseases and asthma, in a mannersimilar to the widespread efficacy of anti-IL-8 antibodies in thetreatment of such disease indications that is known in the art, whichtreatment indications include: (1) ischemic reperfusion injury of thelung (Sekido et.al., Nature, 365: 654 (1993)); (2) acute lung injury andARDS (WO 96/22785 published Aug. 1, 1996; Folkesson et al., J. Clin.Invest., 96: 107–116 (1995); Mulligan et al., J. Immunol., 150:5585–5595 (1993)); (3) hypovolemic shock (Hebert, C., “HumanizedAnti-IL-8: Potential Therapy for Shock and ARDS”, seminar presented atKeystone Conference on The Role of Cytokines in Leukocyte Traffickingand Disease, held at Copper Mountain Resort, Colo., Mar. 31–Apr. 5,1997; Sharar, S. A., Harlan, J. H., Patterson, C. A., Hebert, C. A., andWinn, R. K., “Reperfusion Injury After Hemorrhagic Shock in Rabbits isReduced Similarly by IL-8 or CD-18 Monoclonal Antibodies”, manuscriptsubmitted 1998); (4) myocardial infarction (WO 97/40215 published Oct.30, 1997); (5) cerebral reperfusion injury (Matsumoto et al., LaboratoryInvest., 77: 119–125 (1997)); (6) bacterial pneumonia (U.S. Pat. Nos.5,702,946, 5,677,426, 5,707,622, and 5,686,070); (7) ulcerative colitis(U.S. Pat. Nos. 5,702,946, 5,677,426, 5,707,622, and 5,686,070); andasthma (WO 97/01354 published Jan. 16, 1997).

As shown in the Examples below, the conjugates of the invention mimicthe in vitro activities of full-length anti-IL-8 monoclonal antibody(e.g. inhibition of IL-8 binding and activation of human neutrophils asshown in FIGS. 54A–54C, 55A–55C and 56A–56C and in Example V below),approximate the in vivo pharmacokinetics (e.g. serum half-life,clearance rate and mean residence time as shown in FIG. 65 and inExample X below) and the in vivo therapeutic efficacy (e.g. thetreatment of acute lung injury and ARDS as shown in FIGS. 70A–70E and inExample Z below and the treatment of ischemic reperfusion injury asshown in FIG. 71 and in Example AA below) of full length anti-IL-8monoclonal antibody. Since conjugates of the invention derived fromanti-IL-8 antibodies and fragments display the same or substantiallysimilar in vitro and in vivo activities as full length anti-IL-8monoclonal antibody across a range of different parameters, includingpharmacokinetic characteristics and therapeutic endpoints in variousanimal models, the data support the efficacy of the conjugates in thesame broad spectrum of disease indications that responds to full lengthanti-IL-8 antibody treatment.

As noted above, any conjugate of the invention derived from an anti-IL-8antibody or fragment can be advantageously utilized in a method oftreating an IL-8 mediated disease or disorder, such as inflammatorydiseases. In one embodiment, the invention provides a method of treatingan inflammatory disorder in a mammal comprising administering to themammal an effective amount of a conjugate selected from the groupconsisting of: (1) every conjugate described in Section (II)(1) aboveformed by its component parts, i.e. the antibody fragment or fragmentsand the nonproteinaceous polymer or polymer molecules that form theconjugate, without any extraneous matter in the covalent molecularstructure of the conjugate, (2) every conjugate described in Section(II)(1) above modified to contain one or more additional components, inaddition to the antibody fragment component(s) and polymer component(s)that form the conjugate, wherein the modification does not alter theessential functional property of the conjugate of substantially improvedserum half-life, MRT and/or serum clearance rate as compared to that ofthe parental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating inflammatory disorders wherein at least one antibody fragmentin the conjugate is selected from the group consisting of: (1) anantibody fragment comprising 6G4.2.5LV/L1N35A as defined below; (2) anantibody fragment comprising 6G4.2.5LV/L1N35E as defined below; (3) anantibody fragment comprising 6G4.2.5HV11 as defined below; (4) anantibody fragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) anantibody fragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) anantibody fragment comprising hu6G4.2.5HV as defined below; (7) anantibody fragment comprising 6G4.2.5LV/L1N35A and further comprising theCDRs of 6G4.2.5HV11 as defined below; (8) an antibody fragmentcomprising 6G4.2.5LV/L1N35E and further comprising the CDRs of6G4.2.5HV11 as defined below; (9) an antibody fragment comprisinghu6G4.2.5LV/L1N35A and further comprising hu6G4.2.5HV as defined below;(10) an antibody fragment comprising hu6G4.2.5LV/L1N35E and furthercomprising hu6G4.2.5HV as defined below; (11) an antibody fragmentcomprising 6G4.2.5LV11N35A as defined below; (12) an antibody fragmentcomprising 6G4.2.5LV11N35E as defined below; (13) an antibody fragmentcomprising 6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as definedbelow; and (14) an antibody fragment comprising 6G4.2.5LV11N35E andfurther comprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating an inflammatory disorder wherein the conjugatecontains no more than one antibody fragment, wherein the antibodyfragment is selected from the group consisting of Fab, Fab′ and Fab′-SH,wherein the antibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In another embodiment, the invention provides a method of treatingischemic reperfusion injury in a mammal comprising administering to themammal an effective amount of a conjugate selected from the groupconsisting of: (1) every conjugate described in Section (II)(1) aboveformed by its component parts, i.e. the antibody fragment or fragmentsand the nonproteinaceous polymer or polymer molecules that form theconjugate, without any extraneous matter in the covalent molecularstructure of the conjugate, (2) every conjugate described in Section(II)(1) above modified to contain one or more additional components, inaddition to the antibody fragment component(s) and polymer component(s)that form the conjugate, wherein the modification does not alter theessential functional property of the conjugate of substantially improvedserum half-life, MRT and/or serum clearance rate as compared to that ofthe parental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating ischemic reperfusion injury wherein at least one antibodyfragment in the conjugate is selected from the group consisting of: (1)an antibody fragment comprising 6G4.2.5LV/L1N35A as defined below; (2)an antibody fragment comprising 6G4.2.5LV/L1N35E as defined below; (3)an antibody fragment comprising 6G4.2.5HV11 as defined below; (4) anantibody fragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) anantibody fragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) anantibody fragment comprising hu6G4.2.5HV as defined below; (7) anantibody fragment comprising 6G4.2.5LV/L1N35A and further comprising theCDRs of 6G4.2.5HV11 as defined below; (8) an antibody fragmentcomprising 6G4.2.5LV/L1N35E and further comprising the CDRs of6G4.2.5HV11 as defined below; (9) an antibody fragment comprisinghu6G4.2.5LV/L1N35A and further comprising hu6G4.2.5HV as defined below;(10) an antibody fragment comprising hu6G4.2.5LV/L1N35E and furthercomprising hu6G4.2.5HV as defined below; (11) an antibody fragmentcomprising 6G4.2.5LV11N35A as defined below; (12) an antibody fragmentcomprising 6G4.2.5LV11N35E as defined below; (13) an antibody fragmentcomprising 6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as definedbelow; and (14) an antibody fragment comprising 6G4.2.5LV11N35E andfurther comprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses the foregoing methodsof treating ischemic reperfusion injury wherein the ischemic reperfusioninjury is induced by or incident to a surgical procedure, i.e. asurgical tissue reperfusion injury.

In still another aspect, the invention encompasses the foregoing methodsof treating ischemic reperfusion injury wherein the ischemic reperfusioninjury is a myocardial ischemic reperfusion injury, such as myocardialinfarction, reperfusion after cardiac surgery, cardiac arrest, andconstriction after percutaneous transluminal coronary angioplasty.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating ischemic reperfusion injury wherein the conjugatecontains no more than one antibody fragment, wherein the antibodyfragment is selected from the group consisting of Fab, Fab′ and Fab′-SH,wherein the antibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In another embodiment, the invention provides a method of treating acutelung injury in a mammal comprising administering to the mammal aneffective amount of a conjugate selected from the group consisting of:(1) every conjugate described in Section (II)(1) above formed by itscomponent parts, i.e. the antibody fragment or fragments and thenonproteinaceous polymer or polymer molecules that form the conjugate,without any extraneous matter in the covalent molecular structure of theconjugate, (2) every conjugate described in Section (II)(1) abovemodified to contain one or more additional components, in addition tothe antibody fragment component(s) and polymer component(s) that formthe conjugate, wherein the modification does not alter the essentialfunctional property of the conjugate of substantially improved serumhalf-life, MRT and/or serum clearance rate as compared to that of theparental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating acute lung injury wherein at least one antibody fragment in theconjugate is selected from the group consisting of: (1) an antibodyfragment comprising 6G4.2.5LV/L1N35A as defined below; (2) an antibodyfragment comprising 6G4.2.5LV/L1N35E as defined below; (3) an antibodyfragment comprising 6G4.2.5HV11 as defined below; (4) an antibodyfragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) an antibodyfragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) an antibodyfragment comprising hu6G4.2.5HV as defined below; (7) an antibodyfragment comprising 6G4.2.5LV/L1N35A and further comprising the CDRs of6G4.2.5HV11 as defined below; (8) an antibody fragment comprising6G4.2.5LV/L1N35E and further comprising the CDRs of 6G4.2.5HV11 asdefined below; (9) an antibody fragment comprising hu6G4.2.5LV/L1N35Aand further comprising hu6G4.2.5HV as defined below; (10) an antibodyfragment comprising hu6G4.2.5LV/L1N35E and further comprisinghu6G4.2.5HV as defined below; (11) an antibody fragment comprising6G4.2.5LV11N35A as defined below; (12) an antibody fragment comprising6G4.2.5LV11N35E as defined below; (13) an antibody fragment comprising6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as defined below; and(14) an antibody fragment comprising 6G4.2.5LV11N35E and furthercomprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses the foregoing methodsof treating acute lung injury wherein the acute lung injury includesadult respiratory distress syndrome (ARDS).

In a further aspect, the invention encompasses any of the foregoingmethods of treating acute lung injury wherein the conjugate contains nomore than one antibody fragment, wherein the antibody fragment isselected from the group consisting of Fab, Fab′ and Fab′-SH, wherein theantibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD.

In a further aspect, the invention encompasses any of the foregoingmethods of treating acute lung injury, wherein the patient is selectedfor prophylactic treatment prior to onset of acute lung injury (with orwithout progression to ARDS), such as at least 2 hours prior to onset,or at least 90 minutes prior to onset, or at least 60 minutes prior toonset, or at least 30 minutes prior to onset, by the assessment ofbiological parameters displayed in the patient's condition that indicatelikely progression of disease to acute lung injury which may includeARDS, e.g. by using any of the prognostic methods described in Section(II)(5)(B) below, wherein the conjugate contains no more than oneantibody fragment, wherein the antibody fragment is selected from thegroup consisting of Fab, Fab′ and Fab′-SH, wherein the antibody fragmentis covalently attached to no more than one nonproteinaceous polymermolecule, and wherein the nonproteinaceous polymer molecule is a linearpolyethylene glycol having a molecular weight of at least at or about 20kD, or at least at or about 30 kD or at least at or about 40 kD, or is abranched polyethylene glycol having a molecular weight of at least at orabout 40 kD.

In another embodiment, the invention provides a method of treatinghypovolemic shock in a mammal comprising administering to the mammal aneffective amount of a conjugate selected from the group consisting of:(1) every conjugate described in Section (II)(1) above formed by itscomponent parts, i.e. the antibody fragment or fragments and thenonproteinaceous polymer or polymer molecules that form the conjugate,without any extraneous matter in the covalent molecular structure of theconjugate, (2) every conjugate described in Section (II)(1) abovemodified to contain one or more additional components, in addition tothe antibody fragment component(s) and polymer component(s) that formthe conjugate, wherein the modification does not alter the essentialfunctional property of the conjugate of substantially improved serumhalf-life, MRT and/or serum clearance rate as compared to that of theparental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating hypovolemic shock wherein at least one antibody fragment in theconjugate is selected from the group consisting of: (1) an antibodyfragment comprising 6G4.2.5LV/L1N35A as defined below; (2) an antibodyfragment comprising 6G4.2.5LV/L1N35E as defined below; (3) an antibodyfragment comprising 6G4.2.5HV11 as defined below; (4) an antibodyfragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) an antibodyfragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) an antibodyfragment comprising hu6G4.2.5HV as defined below; (7) an antibodyfragment comprising 6G4.2.5LV/L1N35A and further comprising the CDRs of6G4.2.5HV11 as defined below; (8) an antibody fragment comprising6G4.2.5LV/L1N35E and further comprising the CDRs of 6G4.2.5HV11 asdefined below; (9) an antibody fragment comprising hu6G4.2.5LV/L1N35Aand further comprising hu6G4.2.5HV as defined below; (10) an antibodyfragment comprising hu6G4.2.5LV/L1N35E and further comprisinghu6G4.2.5HV as defined below; (11) an antibody fragment comprising6G4.2.5LV11N35A as defined below; (12) an antibody fragment comprising6G4.2.5LV11N35E as defined below; (13) an antibody fragment comprising6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as defined below; and(14) an antibody fragment comprising 6G4.2.5LV11N35E and furthercomprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating hypovolemic shock wherein the conjugate contains nomore than one antibody fragment, wherein the antibody fragment isselected from the group consisting of Fab, Fab′ and Fab′-SH, wherein theantibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In another embodiment, the invention provides a method of treating aninflammatory bowel disease in a mammal comprising administering to themammal an effective amount of a conjugate selected from the groupconsisting of: (1) every conjugate described in Section (II)(1) aboveformed by its component parts, i.e. the antibody fragment or fragmentsand the nonproteinaceous polymer or polymer molecules that form theconjugate, without any extraneous matter in the covalent molecularstructure of the conjugate, (2) every conjugate described in Section(II)(1) above modified to contain one or more additional components, inaddition to the antibody fragment component(s) and polymer component(s)that form the conjugate, wherein the modification does not alter theessential functional property of the conjugate of substantially improvedserum half-life, MRT and/or serum clearance rate as compared to that ofthe parental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating an inflammatory bowel disease wherein at least one antibodyfragment in the conjugate is selected from the group consisting of: (1)an antibody fragment comprising 6G4.2.5LV/L1N35A as defined below; (2)an antibody fragment comprising 6G4.2.5LV/L1N35E as defined below; (3)an antibody fragment comprising 6G4.2.5HV11 as defined below; (4) anantibody fragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) anantibody fragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) anantibody fragment comprising hu6G4.2.5HV as defined below; (7) anantibody fragment comprising 6G4.2.5LV/L1N35A and further comprising theCDRs of 6G4.2.5HV11 as defined below; (8) an antibody fragmentcomprising 6G4.2.5LV/L1N35E and further comprising the CDRs of6G4.2.5HV11 as defined below; (9) an antibody fragment comprisinghu6G4.2.5LV/L1N35A and further comprising hu6G4.2.5HV as defined below;(10) an antibody fragment comprising hu6G4.2.5LV/L1N35E and furthercomprising hu6G4.2.5HV as defined below; (11) an antibody fragmentcomprising 6G4.2.5LV11N35A as defined below; (12) an antibody fragmentcomprising 6G4.2.5LV11N35E as defined below; (13) an antibody fragmentcomprising 6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as definedbelow; and (14) an antibody fragment comprising 6G4.2.5LV11N35E andfurther comprising 6G4.2.5HV11 as defined below.

In still another aspect, the invention encompasses the foregoing methodsof treating an inflammatory bowel disease wherein the inflammatory boweldisease is ulcerative colitis.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating inflammatory bowel disease wherein the conjugatecontains no more than one antibody fragment, wherein the antibodyfragment is selected from the group consisting of Fab, Fab′ and Fab′-SH,wherein the antibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In another embodiment, the invention provides a method of treating abacterial pneumonia in a mammal comprising administering to the mammalan effective amount of a conjugate selected from the group consistingof: (1) every conjugate described in Section (II)(1) above formed by itscomponent parts, i.e. the antibody fragment or fragments and thenonproteinaceous polymer or polymer molecules that form the conjugate,without any extraneous matter in the covalent molecular structure of theconjugate, (2) every conjugate described in Section (II)(1) abovemodified to contain one or more additional components, in addition tothe antibody fragment component(s) and polymer component(s) that formthe conjugate, wherein the modification does not alter the essentialfunctional property of the conjugate of substantially improved serumhalf-life, MRT and/or serum clearance rate as compared to that of theparental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (I)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating bacterial pneumonia wherein at least one antibody fragment inthe conjugate is selected from the group consisting of: (1) an antibodyfragment comprising 6G4.2.5LV/L1N35A as defined below; (2) an antibodyfragment comprising 6G4.2.5LV/L1N35E as defined below; (3) an antibodyfragment comprising 6G4.2.5HV11 as defined below; (4) an antibodyfragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) an antibodyfragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) an antibodyfragment comprising hu6G4.2.5HV as defined below; (7) an antibodyfragment comprising 6G4.2.5LV/L1N35A and further comprising the CDRs of6G4.2.5HV11 as defined below; (8) an antibody fragment comprising6G4.2.5LV/L1N35E and further comprising the CDRs of 6G4.2.5HV11 asdefined below; (9) an antibody fragment comprising hu6G4.2.5LV/L1N35Aand further comprising hu6G4.2.5HV as defined below; (10) an antibodyfragment comprising hu6G4.2.5LV/L1N35E and further comprisinghu6G4.2.5HV as defined below; (11) an antibody fragment comprising6G4.2.5LV11N35A as defined below; (12) an antibody fragment comprising6G4.2.5LV11N35E as defined below; (13) an antibody fragment comprising6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as defined below; and(14) an antibody fragment comprising 6G4.2.5LV11N35E and furthercomprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating bacterial pneumonia wherein the conjugate containsno more than one antibody fragment, wherein the antibody fragment isselected from the group consisting of Fab, Fab′ and Fab′-SH, wherein theantibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In another embodiment, the invention provides a method of treating anasthmatic disease in a mammal comprising administering to the mammal aneffective amount of a conjugate selected from the group consisting of:(1) every conjugate described in Section (II)(1) above formed by itscomponent parts, i.e. the antibody fragment or fragments and thenonproteinaceous polymer or polymer molecules that form the conjugate,without any extraneous matter in the covalent molecular structure of theconjugate, (2) every conjugate described in Section (II)(1) abovemodified to contain one or more additional components, in addition tothe antibody fragment component(s) and polymer component(s) that formthe conjugate, wherein the modification does not alter the essentialfunctional property of the conjugate of substantially improved serumhalf-life, MRT and/or serum clearance rate as compared to that of theparental antibody fragment from which the conjugate is derived, (3)every conjugate described in Section (II)(1) above modified toincorporate one or more nonproteinaceous labels or reporter molecules,and (4) every conjugate described in Section (II)(1) above modified toincorporate one or more radiolabels; wherein at least one antibodyfragment in the conjugate comprises an antigen binding site that bindsto human IL-8.

In another aspect, the invention encompasses the foregoing method oftreating an asthmatic disease wherein at least one antibody fragment inthe conjugate is selected from the group consisting of: (1) an antibodyfragment comprising 6G4.2.5LV/L1N35A as defined below, (2) an antibodyfragment comprising 6G4.2.5LV/L1N35E as defined below; (3) an antibodyfragment comprising 6G4.2.5HV11 as defined below; (4) an antibodyfragment comprising hu6G4.2.5LV/L1N35A as defined below; (5) an antibodyfragment comprising hu6G4.2.5LV/L1N35E as defined below; (6) an antibodyfragment comprising hu6G4.2.5HV as defined below; (7) an antibodyfragment comprising 6G4.2.5LV/L1N35A and further comprising the CDRs of6G4.2.5HV11 as defined below; (8) an antibody fragment comprising6G4.2.5LV/L1N35E and further comprising the CDRs of 6G4.2.5HV11 asdefined below; (9) an antibody fragment comprising hu6G4.2.5LV/L1N35Aand further comprising hu6G4.2.5HV as defined below; (10) an antibodyfragment comprising hu6G4.2.5LV/L1N35E and further comprisinghu6G4.2.5HV as defined below; (11) an antibody fragment comprising6G4.2.5LV11N35A as defined below; (12) an antibody fragment comprising6G4.2.5LV11N35E as defined below; (13) an antibody fragment comprising6G4.2.5LV11N35A and further comprising 6G4.2.5HV11 as defined below; and(14) an antibody fragment comprising 6G4.2.5LV11N35E and furthercomprising 6G4.2.5HV11 as defined below.

In yet another aspect, the invention encompasses the foregoing methodsof treating asthmatic disease wherein the asthmatic disease is allergicasthma.

In yet another aspect, the invention encompasses any of the foregoingmethods of treating an asthmatic disease wherein the conjugate containsno more than one antibody fragment, wherein the antibody fragment isselected from the group consisting of Fab, Fab′ and Fab′-SH, wherein theantibody fragment is covalently attached to no more than onenonproteinaceous polymer molecule, and wherein the nonproteinaceouspolymer molecule is a linear polyethylene glycol having a molecularweight of at least at or about 20 kD, or at least at or about 30 kD orat least at or about 40 kD, or is a branched polyethylene glycol havinga molecular weight of at least at or about 40 kD.

In a preferred embodiment, the invention encompasses any of theforegoing methods of treating inflammatory diseases or asthmaticdiseases wherein the mammal is a human.

Therapeutic formulations of the conjugate of the invention can beprepared by utilizing the same procedures described for the formulationof the anti-IL-8 antibodies and fragments of the invention in Section(II)(5)(B) below. The conjugate of the invention can be administered inplace of the parent antibody for a given disease indication by modifyingthe formulation, dosage, administration protocol, and other aspects of atherapeutic regimen as required by the different pharmacodynamiccharacteristics of the conjugate and as dictated by common medicalknowledge and practice.

e. Reagent Uses for Large Effective Size Conjugates

The conjugate of the invention also finds application as a reagent in ananimal model system for in vivo study of the biological functions of theantigen recognized by the conjugate.

The conjugate would enable the practitioner to inactivate or detect thecognate antigen in circulation or in tissue for a far greater period oftime than would be possible with art-known constructs while removing anyFc interaction (which could attend the use of an intact antibody) fromthe system. In addition, the increased half-life of the conjugate of theinvention can be applied advantageously to the induction of tolerancefor the underivatized antibody fragment in a test animal by employingthe Wie et al., Int. Archs. Allergy Appl. Immunol. 64: 84–99 (1981)method for allergen tolerization, which would permit the practitioner torepeatedly challenge the tolerized animal with the underivatizedparental antibody fragment without generating an immune response againstthe parental fragment.

2. Humanized 6G4.2.5 Monoclonal Antibodies and Antibody Fragments

In one embodiment, the invention provides an antibody fragment or fulllength antibody comprising a heavy chain comprising the amino acidsequence of amino acids 1–230 (herein referred to as “6G4.2.5HV11”) ofthe humanized anti-IL-8 6G4.2.5v11 heavy chain polypeptide amino acidsequence of FIGS. 37A–37B (SEQ ID NO: 60).

The invention encompasses a single chain antibody fragment comprisingthe 6G4.2.5HV11, with or without any additional amino acid sequence. Inone embodiment, the invention provides a single chain antibody fragmentcomprising the 6G4.2.5HV11 without any associated light chain amino acidsequence, i.e. a single chain species that makes up one half of a Fabfragment.

Further provided herein are an antibody or antibody fragment comprisingthe 6G4.2.5HV11, and further comprising a light chain comprising theamino acid sequence of amino acids 1–219 (herein referred to as“6G4.2.5LV11”) of the humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51).

In one embodiment, the invention provides a single chain antibodyfragment wherein the 6G4.2.5HV11 and the 6G4.2.5LV11 are contained in asingle chain polypeptide species. In a preferred embodiment, the singlechain antibody fragment comprises the 6G4.2.5HV11 joined to the6G4.2.5LV11 by means of a flexible peptide linker sequence, wherein theheavy chain and light chain domains can associate in a “dimeric”structure analogous to that formed in a two-chain Fab species. Inanother embodiment, the single chain antibody fragment is a speciescomprising the 6G4.2.5HV11 joined to the 6G4.2.5LV11 by a linker that istoo short to permit intramolecular pairing of complementary domains,i.e. a single chain polypeptide monomer that forms a diabody upondimerization with another monomer.

In yet another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the 6G4.2.5HV11 and a second polypeptide chain comprisesthe 6G4.2.5LV11 and the two polypeptide chains are covalently linked byone or more interchain disulfide bonds. In a preferred embodiment, theforegoing two-chain antibody fragment is selected from the groupconsisting of Fab, Fab′, Fab′-SH, and F(ab′)₂.

The invention also provides an antibody or antibody fragment comprisinga heavy chain containing the 6G4.2.5HV11 and optionally furthercomprising a light chain containing the 6G4.2.5LV11, wherein the heavychain, and optionally the light chain, is (are) fused to an additionalmoiety, such as additional immunoglobulin constant domain sequence.Constant domain sequence can be added to the heavy chain and/or lightchain sequence(s) to form species with full or partial length heavyand/or light chain(s). It will be appreciated that constant regions ofany isotype can be used for this purpose, including IgG, IgM, IgA, IgD,and IgE constant regions, and that such constant regions can be obtainedfrom any human or animal species. Preferably, the constant domainsequence is human in origin. Suitable human constant domain sequencescan be obtained from Kabat et al. (supra).

In a preferred embodiment, the antibody or antibody fragment comprisesthe 6G4.2.5HV11 in a heavy chain that is fused to or contains a leucinezipper sequence. The leucine zipper can increase the affinity and/orproduction efficiency of the antibody or antibody fragment of interest.Suitable leucine zipper sequences include the jun and fos leucinezippers taught by Kostelney et al., J. Immunol., 148: 1547–1553 (1992)and the GCN4 leucine zipper described in the Examples below. In apreferred embodiment, the antibody or antibody fragment comprises the6G4.2.5HV11 fused at its C-terminus to the GCN4 leucine zipper to yieldthe amino acid sequence of amino acids 1–275 (herein referred to as“6G4.2.5HV11GCN4”) of the heavy chain polypeptide amino acid sequence ofFIGS. 37A–37B (SEQ ID NO: 60).

3. Variants of Humanized 6G4.2.5 Monoclonal Antibodies and AntibodyFragments

The invention additionally encompasses humanized anti-IL-8 monoclonalantibody and antibody fragments comprising variants of the 6G4.2.5complementarity determining regions (CDRs) or variants of the 6G4.2.5v11variable domains which exhibit higher affinity for human IL-8 and/orpossess properties that yield greater efficiency in recombinantproduction processes.

A. 6G4.2.5LV Variants

In one aspect, the invention provides humanized anti-IL-8 monoclonalantibodies and antibody fragments comprising the complementaritydetermining regions (referred to herein as the “CDRs of 6G4.2.5LV”) L1,L2, and L3 of the 6G4.2.5 light chain variable domain amino acidsequence of FIG. 24, wherein L1 corresponds to amino acids 24–39 of theamino acid sequence of FIG. 24, L2 corresponds to amino acids 55–61 ofthe amino acid sequence of FIG. 24 (SEQ ID NO: 35), and L3 correspondsto amino acids 94–102 of the amino acid sequence of FIG. 24 (SEQ ID NO:35).

In addition, the invention provides a variant 6G4.2.5 humanized antibodyor antibody fragment comprising a humanized light chain variable domaincomprising a variant (hereinafter referred to a “6G4.2.5LV CDRsvariant”) of the complementarity determining regions L1, L2, and L3 ofthe 6G4.2.5 variable light chain domain amino acid sequence of FIG. 24(SEQ ID NO: 35). In one embodiment, the invention provides a variant6G4.2.5 humanized antibody or antibody fragment comprising a 6G4.2.5LVCDRs variant (herein referred to as “6G4.2.5LV/L1N35X₃₅”) wherein L1corresponds to amino acids 24–39 of the amino acid sequence of FIG. 24(SEQ ID NO: 35) with the proviso that any amino acid other than Asn(denoted as “X₃₅”) is substituted for Asn at amino acid position 35, L2corresponds to amino acids 55–61 of the amino acid sequence of FIG. 24(SEQ ID NO: 35), and L3 corresponds to amino acids 94–102 of the aminoacid sequence of FIG. 24 (SEQ ID NO: 35). In a preferred embodiment, theinvention provides a variant 6G4.2.5 humanized antibody or antibodyfragment comprising a 6G4.2.5LV CDRs variant (herein referred to as“6G4.2.5LV/L1N35A”) wherein L1 corresponds to amino acids 24–39 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35) with the proviso that Alais substituted for Asn at amino acid position 35, L2 corresponds toamino acids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35),and L3 corresponds to amino acids 94–102 of the amino acid sequence ofFIG. 24 (SEQ ID NO: 35). In another preferred embodiment, the inventionprovides a variant 6G4.2.5 humanized antibody or antibody fragmentcomprising a 6G4.2.5LV CDRs variant (herein referred to as“6G4.2.5LV/L1N35E”) wherein L1 corresponds to amino acids 24–39 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35) with the proviso that Gluis substituted for Asn at amino acid position 35, L2 corresponds toamino acids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35),and L3 corresponds to amino acids 94–102 of the amino acid sequence ofFIG. 24 (SEQ ID NO: 35).

In a second aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L1S26X₂₆”) wherein L1 corresponds toamino acids 24–39 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35)with the proviso that any amino acid other than Ser (denoted as “X₂₆”)is substituted for Ser at amino acid position 26, L2 corresponds toamino acids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35),and L3 corresponds to amino acids 94–102 of the amino acid sequence ofFIG. 24 (SEQ ID NO: 35). In a preferred embodiment, the inventionprovides a variant 6G4.2.5 humanized antibody or antibody fragmentcomprising a 6G4.2.5LV CDRs variant (herein referred to as“6G4.2.5LV/L1S26A”) wherein L1 corresponds to amino acids 24–39 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35) with the proviso that Alais substituted for Ser at amino acid position 26, L2 corresponds toamino acids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35),and L3 corresponds to amino acids 94–102 of the amino acid sequence ofFIG. 24 (SEQ ID NO: 35).

In a third aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L3H98X₉₈”) wherein L1 corresponds toamino acids 24–39 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35),L2 corresponds to amino acids 55–61 of the amino acid sequence of FIG.24 (SEQ ID NO: 35), and L3 corresponds to amino acids 94–102 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35) with the proviso that anyamino acid other than His (denoted as “X₉₈”) is substituted for His atamino acid position 98. In a preferred embodiment, the inventionprovides a variant 6G4.2.5 humanized antibody or antibody fragmentcomprising a 6G4.2.5LV CDRs variant (herein referred to as“6G4.2.5LV/L3H98A”) wherein L1 corresponds to amino acids 24–39 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35), L2 corresponds to aminoacids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35), andL3 corresponds to amino acids 94–102 of the amino acid sequence of FIG.24 (SEQ ID NO: 35) with the proviso that Ala is substituted for His atamino acid position 98.

In a fourth aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L1S26X₂₆,N35X₃₅”) wherein L1corresponds to amino acids 24–39 of sequence of FIG. 24 (SEQ ID NO: 35)with the proviso that any amino acid other than Ser (denoted as “X₂₆”)is substituted for Ser at amino acid position 26 and any amino acidother than Asn (denoted as “X₃₅”) is substituted for Asn at amino acidposition 35, L2 corresponds to amino acids 55–61 of the amino acidsequence of FIG. 24 (SEQ ID NO:35), and L3 corresponds to amino acids94–102 of the amino acid sequence of FIG. 24 (SEQ ID NO:35). In apreferred embodiment, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L1S26A,N35A”) wherein L1 correspondsto amino acids 24–39 of the amino acid sequence of FIG. 24 (SEQ IDNO:35) with the proviso that Ala is substituted for Ser at amino acidposition 26 and Ala is substituted for Asn at amino acid position 35, L2corresponds to amino acids 55–61 of the amino acid sequence of FIG. 24(SEQ ID NO 35), and L3 corresponds to amino acids 94–102 of the aminoacid sequence of FIG. 24 (SEQ ID NO 35).

In a fifth aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L1N35X₃₅/L3H98X₉₈”) wherein L1corresponds to amino acids 24–39 of the amino acid sequence of FIG. 24(SEQ ID NO 35) with the proviso that any amino acid other than Asn(denoted as “X₃₅”) is substituted for Asn at amino acid position 35, L2corresponds to amino acids 55–61 of the amino acid sequence of FIG. 24(SEQ ID NO 35), and L3 corresponds to amino acids 94–102 of the aminoacid sequence of FIG. 24 (SEQ ID NO 35) with the proviso that any aminoacid other than His (denoted as “X₉₈”) is substituted for His at aminoacid position 98. In a preferred embodiment, the invention provides avariant 6G4.2.5 humanized antibody or antibody fragment comprising a6G4.2.5LV CDRs variant (herein referred to as “6G4.2.5LV/L1N35A/L3H98A”)wherein L1 corresponds to amino acids 24–39 of the amino acid sequenceof FIG. 24 (SEQ ID NO 35) with the proviso that Ala is substituted forAsn at amino acid position 35, L2 corresponds to amino acids 55–61 ofthe amino acid sequence of FIG. 24 (SEQ ID NO 35), and L3 corresponds toamino acids 94–102 of the amino acid sequence of FIG. 24 (SEQ ID NO 35)with the proviso that Ala is substituted for His at amino acid position98.

In a sixth aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant(herein referred to as “6G4.2.5LV/L1S26X₂₆/L3H98X₈₉”) wherein L1corresponds to amino acids 24–39 of the amino acid sequence of FIG. 24(SEQ ID NO 35) with the proviso that any amino acid other than Ser(denoted as “X₂₆”) is substituted for Ser at amino acid position 26, L2corresponds to amino acids 55–61 of the amino acid sequence of FIG. 24(SEQ ID NO 35), and L3 corresponds to amino acids 94–102 of the aminoacid sequence of FIG. 24 (SEQ ID NO 35) with the proviso that any aminoacid other than His (denoted as “X₉₈”) is substituted for His at aminoacid position 98. In a preferred embodiment, the invention provides avariant 6G4.2.5 humanized antibody or antibody fragment comprising a6G4.2.5LV CDRs variant (herein referred to as “6G4.2.5LV/L1S26A/L3H98A”)wherein L1 corresponds to amino acids 24–39 of the amino acid sequenceof FIG. 24 (SEQ ID NO 35) with the proviso that Ala is substituted forSer at amino acid position 26, L2 corresponds to amino acids 55–61 ofthe amino acid sequence of FIG. 24 SEQ ID NO 35), and L3 corresponds toamino acids 94–102 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35)with the proviso that Ala is substituted for His at amino acid position98.

In a seventh aspect, the invention provides a variant 6G4.2.5 humanizedantibody or antibody fragment comprising a 6G4.2.5LV CDRs variant (herereferred to as “6G4.2.5LV/L1S26X₂₆,N35X₃₅/L3H98X₉₈”) wherein L1corresponds to amino acids 24–39 of the amino acid sequence of FIG. 24(SEQ ID NO: 35) with the proviso that any amino acid other than Ser(denoted as “X₂₆”) is substituted for Ser at amino acid position 26 andany amino acid other than Asn (denoted as “X₃₅”) is substituted for Asnat amino acid position 35, L2 corresponds to amino acids 55–61 of theamino acid sequence of FIG. 24 (SEQ ID NO: 35), and L3 corresponds toamino acids 94–102 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35)with the proviso that any amino acid other than His (denoted as “X₉₈”)is substituted for His at amino acid position 98. In a preferredembodiment, the invention provides a variant 6G4.2.5 humanized antibodyor antibody fragment comprising a 6G4.2.5LV CDRs variant (here referredto as “6G4.2.5LV/L1S26A,N35A/L3H98A”) wherein L1 corresponds to aminoacids 24–39 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35) withthe proviso that Ala is substituted for Ser at amino acid position 26and Ala is substituted for Asn at amino acid position 35, L2 correspondsto amino acids 55–61 of the amino acid sequence of FIG. 24 (SEQ ID NO:35), and L3 corresponds to amino acids 94–102 of the amino acid sequenceof FIG. 24 (SEQ ID NO: 35) with the proviso that Ala is substituted forHis at amino acid position 98.

The humanized light chain variable domains of the invention can beconstructed by using any of the techniques for antibody humanizationknown in the art. Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522(1986); Riechmann et al., Nature 332:323 (1988); Verboeyen et al.,Science 239:1534 (1988)), by substituting the CDRs of 6G4.2.5LV or theCDRs of a 6G4.2.5LV CDRs variant for the corresponding sequences of ahuman antibody light chain variable domain. Accordingly, such“humanized” derivatives containing the CDRs of 6G4.2.5LV or the CDRs ofa 6G4.2.5VL CDRs variant are chimeric (Cabilly et al., supra). Thehumanized light chain variable domain comprising the CDRs of 6G4.2.5LVor the CDRs of a 6G4.2.5LV CDRs variant can also contain some FRresidues that are substituted by residues from analogous sites in themurine 6G4.2.5 antibody light chain variable domain (“6G4.2.5LV”). Thecomplete amino acid sequence of 6G4.2.5LV is set out as amino acids1–114 of the amino acid sequence of FIG. 24 (SEQ ID NO: 35).

The invention further provides a humanized antibody or antibody fragmentcomprising a humanized light chain variable domain comprising the CDRsof 6G4.2.5LV or the CDRs of a 6G4.2.5LV CDRs variant as described above,and further comprising a humanized heavy chain variable domaincomprising the complementarity determining regions (CDRs) H1, H2, and H3of the 6G4.2.5 (murine monoclonal antibody) variable heavy chain domainamino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H1 correspond toamino acids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),wherein H2 corresponds to amino acids 50–66 of the amino acid sequenceof FIG. 25 (SEQ ID NO: 37), and wherein H3 corresponds to amino acids99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37). Theabove-described H1, H2, and H3CDRs of the 6G4.2.5 heavy chain variabledomain (“6G4.2.5HV”) are collectively referred to as the “CDRs of6G4.2.5HV”.

In another embodiment, the invention provides a humanized antibody orantibody fragment comprising a humanized light chain variable domaincomprising the CDRs of 6G4.2.5LV or the CDRs of a 6G4.2.5LV CDRs variantas described above, and further comprising a humanized heavy chainvariable domain comprising a variant (herein referred to as a “6G4.2.5HVCDRs variant”) of the H1, H2, and H3CDRs of the 6G4.2.5 (murinemonoclonal antibody) variable heavy chain domain amino acid sequence ofFIG. 25 (SEQ ID NO: 37). In one 6G4.2.5HV CDRs variant (referred toherein as “6G4.2.5HV/H1S31Z₃₁”), H1 correspond to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso thatany amino acid other than Ser (denoted as “Z₃₁”) is substituted for Serat amino acid position 31, H2 corresponds to amino acids 50–66 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37). In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A”), H1 correspond to amino acids 26–35 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Ala issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), andH3 corresponds to amino acids 99–111 of the amino acid sequence of FIG.25 (SEQ ID NO: 37).

In a second 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄”), H1 corresponds to amino acids 26–35 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds to amino acids50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37). In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A”), H1 corresponds to amino acids 26–35 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds to amino acids50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Ala is substituted for Ser at amino acid position 54, andH3 corresponds to amino acids 99–111 of the amino acid sequence of FIG.25 (SEQ ID NO: 37).

In a third 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3D100E”), wherein H1 correspond to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2 correspondsto amino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO:37), and wherein H3 corresponds to amino acids 99–111 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Glu issubstituted for Asp at amino acid position 100.

In a fourth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3R102K”), wherein H1 correspond to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2 correspondsto amino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO:37), and wherein H3 corresponds to amino acids 99–111 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Lys issubstituted for Arg at amino acid position 102.

In a fifth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3D106E”), wherein H1 correspond to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2 correspondsto amino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO:37), and wherein H3 corresponds to amino acids 99–111 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Glu issubstituted for Asp at amino acid position 106.

In a seventh 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3D100E,R102K”), wherein H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37), and wherein H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100 and Lys is substitutedfor Arg at amino acid position 102.

In an eighth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3R102K,D106E”), wherein H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37), and wherein H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Lysis substituted for Arg at amino acid position 102 and Glu is substitutedfor Asp at amino acid position 106.

In a ninth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3D100E,D106E”), wherein H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37), and wherein H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100 and Glu is substitutedfor Asp at amino acid position 106.

In a tenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H3D100E,R102K,D106E”), wherein H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), wherein H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37), and wherein H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100, Lys is substitutedfor Arg at amino acid position 102, and Glu is substituted for Asp atamino acid position 106.

In an eleventh 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄”), H1 correspond to amino acids 26–35 ofsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that any amino acidother than Ser (denoted as “Z₃₁”) is substituted for Ser at amino acidposition 31, H2 corresponds to amino acids 50–66 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that any amino acidother than Ser (denoted as “Z₅₄”) is substituted for Ser at amino acidposition 54, and H3 corresponds to amino acids 99–111 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37). In a preferred 6G4.2.5HV CDRsvariant (referred to herein as “6G4.2.5HV/H1S31A/H2S54A”), H1 correspondto amino acids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO:37) with the proviso that Ala is substituted for Ser at amino acidposition 31, H2 corresponds to amino acids 50–66 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Ala issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37).

In a twelfth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3D100E”), H1 correspond to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that anyamino acid other than Ser (denoted as “Z₃₁”) is substituted for Ser atamino acid position 31, H2 corresponds to amino acids 50–66 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37), and H3 corresponds to aminoacids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that Glu is substituted for Asp at amino acid position 100.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3D100E”), H1 correspond to amino acids 26–35 theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Alais substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 100.

In a thirteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃/H3R102K”), H1 correspond to amino acids 26–35 of theamin sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that any aminoacid other than Ser (denoted as “Z₃₁”) is substituted for Ser at aminoacid position 31, H2 corresponds to amino acids 50–66 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37), and H3 corresponds to amino acids99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Lys is substituted for Arg at amino acid position 102. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3R102K”), H1 correspond to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Alais substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Lys is substituted for Argat amino acid position 102.

A fourteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3D106E”), H1 correspond to amino acids 26–35 of theami sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that any aminoacid other than Ser (denoted as “Z₃₁”) is substituted for Ser at aminoacid position 31, H2 corresponds to amino acids 50–66 of the amino acidsequence of FIG. 25 (SEQ ID NO: 37), and H3 corresponds to amino acids99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 0.37) with theproviso that Glu is substituted for Asp at amino acid position 106. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3D106E”), H1 correspond to amino acids 26–35 theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Alais substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 106.

A fifteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat any amino acid other than Ser (denoted as “Z₃₁”) is substituted forSer at amino acid position 31, H2 corresponds to amino acids 50–66 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), and H3 correspondsto amino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37) with the proviso that Glu is substituted for Asp at amino acidposition 100 and Lys is substituted for Arg at amino acid position 102.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3D100E,R102K”), H1 correspond to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso thatAla is substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 100 and Lys is substituted for Arg at amino acidposition 102.

In a sixteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3R102K,D106E”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat any amino acid other than Ser (denoted as “Z₃₁”) is substituted forSer at amino acid position 31, H2 corresponds to amino acids 50–66 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), and H3 correspondsto amino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37) with the proviso that Lys is substituted for Arg at amino acidposition 102 and Glu is substituted for Asp at amino acid position 106.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3R102K,D106E”), H1 correspond to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso thatAla is substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Lys is substituted for Argat amino acid position 102 and Glu is substituted for Asp at amino acidposition 106.

In a seventeenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3D100E,D106E”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat any amino acid other than Ser (denoted as “Z₃₁”) is substituted forSer at amino acid position 31, H2 corresponds to amino acids 50–66 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), and H3 correspondsto amino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO:37) with the proviso that Glu is substituted for Asp at amino acidposition 100 and Glu is substituted for Asp at amino acid position 106.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3D100E,D106E”), H1 correspond to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso thatAla is substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 100 and Glu is substituted for Asp at amino acidposition 106.

In an eighteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K,D106E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, and H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37),and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 100, Lys is substituted for Arg at amino acidposition 102 and Glu is substituted for Asp at amino acid position 106.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H3D100E,R102K,D106E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37), and H3 corresponds to amino acids 99–111 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Glu issubstituted for Asp at amino acid position 100, Lys is substituted forArg at amino acid position 102 and Glu is substituted for Asp at aminoacid position 106.

In a nineteenth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3D100E”), H1 corresponds to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3D100E”), H1 corresponds to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that Ala is substituted for Ser at amino acid position 54,and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 100.

In a twentieth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3R102K”), H1 corresponds to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Lys is substituted for Arg at amino acid position102. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3R102K”), H1 corresponds to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that Ala is substituted for Ser at amino acid position 54,and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Lys is substituted for Argat amino acid position 102.

In a twenty-first 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3D106E”), H1 corresponds to amino acids 26–35 ofthe amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position106. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3D106E”), H1 corresponds to amino acids 26–35 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that Ala is substituted for Ser at amino acid position 54,and H3 corresponds to amino acids 99–111 of the amino acid sequence ofFIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Aspat amino acid position 106.

In a twenty-second 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3D100E,R102K”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100 and Lys is substituted for Arg at amino acid position 102. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3D100E,R102K”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Ala is substituted for Ser at amino acid position54, and H3 corresponds to amino acids 99–111 of the amino acid sequenceof FIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted forAsp at amino acid position 100 and Lys is substituted for Arg at aminoacid position 102.

In a twenty-third 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3R102K,D106E”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Lys is substituted for Arg at amino acid position102 and Glu is substituted for Asp at amino acid position 106. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3R102K,D106E”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Ala is substituted for Ser at amino acid position54, and H3 corresponds to amino acids 99–111 of the amino acid sequenceof FIG. 25 (SEQ ID NO: 37) with the proviso that Lys is substituted forArg at amino acid position 102 and Glu is substituted for Asp at aminoacid position 106.

In a twenty-fourth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3D100E,D106E”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100 and Glu is substituted for Asp at amino acid position 106. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3D100E,D106E”), H1 corresponds to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Ala is substituted for Ser at amino acid position54, and H3 corresponds to amino acids 99–111 of the amino acid sequenceof FIG. 25 (SEQ ID NO: 37) with the proviso that Glu is substituted forAsp at amino acid position 100 and Glu is substituted for Asp at aminoacid position 106.

In a twenty-fifth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54Z₅₄/H3D100E,R102K,D106E”), H1 corresponds to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that any amino acid other than Ser(denoted as “Z₅₄”) is substituted for Ser at amino acid position 54, andH3 corresponds to amino acids 99–111 of the amino acid sequence of FIG.25 (SEQ ID NO: 37) with the proviso that Glu is substituted for Asp atamino acid position 100, Lys is substituted for Arg at amino acidposition 102 and Glu is substituted for Asp at amino acid position 106.In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H2S54A/H3D100E,R102K,D106E”), H1 corresponds to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37), H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100, Lys is substitutedfor Arg at amino acid position 102 and Glu is substituted for Asp atamino acid position 106.

In a twenty-sixth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3D100E”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100.

In a twenty-seventh 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Lys is substituted for Arg at amino acid position102. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3R102K”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Lysis substituted for Arg at amino acid position 102.

In a twenty-eighth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D106E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position106. In a preferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3D106E”), H1 correspond to amino acids 26–35of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with the provisothat Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 106.

In a twenty-ninth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K”), H1 correspond to aminoacids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100 and Lys is substituted for Arg at amino acid position 102. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100 and Lys is substitutedfor Arg at amino acid position 102.

In a thirtieth 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K,D106E”), H1 correspond to aminoacids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Lys is substituted for Arg at amino acid position102 and Glu is substituted for Asp at amino acid position 106. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3R102K,D106E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Lysis substituted for Arg at amino acid position 102 and Glu is substitutedfor Asp at amino acid position 106.

In a thirty-first 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,D106E”), H1 correspond to aminoacids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₃₁”) issubstituted for Ser at amino acid position 31, H2 corresponds to aminoacids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that any amino acid other than Ser (denoted as “Z₅₄”) issubstituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100 and Glu is substituted for Asp at amino acid position 106. In apreferred 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3D100E,D106E”), H1 correspond to amino acids26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) with theproviso that Ala is substituted for Ser at amino acid position 31, H2corresponds to amino acids 50–66 of the amino acid sequence of FIG. 25(SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100 and Glu is substitutedfor Asp at amino acid position 106.

In a thirty-second 6G4.2.5HV CDRs variant (referred to herein as“6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K,D106E”), H1 correspond toamino acids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₃₁”)is substituted for Ser at amino acid position 31, H2 corresponds toamino acids 50–66 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that any amino acid other than Ser (denoted as “Z₅₄”)is substituted for Ser at amino acid position 54, and H3 corresponds toamino acids 99–111 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37)with the proviso that Glu is substituted for Asp at amino acid position100, Lys is substituted for Arg at amino acid position 102 and Glu issubstituted for Asp at amino acid position 106. In a preferred 6G4.2.5HVCDRs variant (referred to herein as“6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K,D106E”), H1 correspond to aminoacids 26–35 of the amino acid sequence of FIG. 25 (SEQ ID NO: 37) withthe proviso that Ala is substituted for Ser at amino acid position 31,H2 corresponds to amino acids 50–66 of the amino acid sequence of FIG.25 (SEQ ID NO: 37) with the proviso that Ala is substituted for Ser atamino acid position 54, and H3 corresponds to amino acids 99–111 of theamino acid sequence of FIG. 25 (SEQ ID NO: 37) with the proviso that Gluis substituted for Asp at amino acid position 100, Lys is substitutedfor Arg at amino acid position 102 and Glu is substituted for Asp atamino acid position 106.

As in the humanization of the light chain variable domain describedabove, a humanized heavy chain variable domain is constructed bysubstituting the CDRs of 6G4.2.5HV or the CDRs of a 6G4.2.5HV CDRsvariant for the corresponding sequences in a human heavy chain variabledomain. The humanized heavy chain variable domain comprising the CDRs of6G4.2.5HV or the CDRs of a 6G4.2.5HV CDRs variant can also contain someFR residues that are substituted by residues from analogous sites in themurine 6G4.2.5 antibody heavy chain variable domain. The complete aminoacid sequence of 6G4.2.5HV is set out as amino acids 1–122 of the aminoacid sequence of FIG. 25 (SEQ ID NO: 37).

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies and antibody fragments is veryimportant to reduce antigenicity. According to the so-called “best-fit”method, the sequence of the variable domain of a rodent antibody isscreened against the entire library of known human variable-domainsequences. The human sequence which is closest to that of the rodent isthen accepted as the human framework (FR) for the humanized antibody(Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987)). Another method uses a particular frameworkderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework can beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993)).

It is also important that the antibodies and antibody fragments of theinvention be humanized with retention of high affinity for human IL-8and other favorable biological properties. To achieve this goal,according to a preferred method, the humanized antibodies and antibodyfragments of the invention are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and parental sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV are collectively referred to hereinas “hu6G4.2.5LV”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1N35X₃₅ are collectively referredto herein as “hu 6G4.2.5LV/L1N35X₃₅”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1N35A are collectively referred toherein as “hu6G4.2.5LV/L1N35A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1N35E are collectively referred toherein as “hu6G4.2.5LV/L1N35E”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26X₂₆ are collectively referredto herein as “hu6G4.2.5LV/L1S26X₂₆”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26A are collectively referred toherein as “hu6G4.2.5LV/L1S26A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L3H98X₉₈ are collectively referredto herein as “hu6G4.2.5LV/L3H98X₉₈”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L3H98A are collectively referred toherein as “hu6G4.2.5LV/L3H98A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26X₂₆,N35X₃₅ are collectivelyreferred to herein as “hu6G4.2.5LV/L1S26X₂₆,N35X₃₅”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26A,N35A are collectivelyreferred to herein as “hu6G4.2.5LV/L1S26A,N35A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1N35X₃₅/L3H98X₉₈ are collectivelyreferred to herein as “hu6G4.2.5LV/L1N35X₃₅/L3H98X₉₈”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1N35A/L3H98A are collectivelyreferred to herein as “hu6G4.2.5LV/L1N35A/L3H98A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26X₂₆/L3H98X₉₈ are collectivelyreferred to herein as “hu6G4.2.5LV/L1S26X₂₆/L3H98X₉₈”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26A/L3H98A are collectivelyreferred to herein as “hu6G4.2.5LV/L1S26A/L3H98A”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26X₂₆,N35X₃₅/L3H98X₉₈ arecollectively referred to herein as“hu6G4.2.5LV/L1S26X₂₆,N35X₃₅/L3H98X₉₈”.

Any and all humanized light chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5LV/L1S26A,N35A/L3H98A are collectivelyreferred to herein as “hu6G4.2.5LV/L1S26A,N35A/L3H98A”.

The humanized light chain variable domain amino acid sequences ofhu6G4.2.5LV/L1N35X₃₅, hu6G4.2.5LV/L1S26X₂₆, hu6G4.2.5LV/L1S26X₂₆/L3H98Xhu6G4.2.5LV/L1S26X₂₆,N35X₃₅, hu6G4.2.5LV/L1N35X₃₅/L3H98X₉₈,hu6G4.2.5LV/L1S26X₂₆/L3H98X₉₈, and hu6G4.2.5LV/L1S26X₂₆,N35X₃₅/L3H98X₈are collectively referred to herein as “hu6G4.2.5LV/vL1-3X”.

The humanized light chain variable domain amino acid sequences ofhu6G4.2.5LV/L1N35A, hu6G4.2.5LV/L1S26A, hu6G4.2.5LV/L1S26A/L3H98A,hu6G4.2.5LV/L1S26A,N35A, hu6G4.2.5LV/L1N35A/L3H98A,hu6G4.2.5LV/L1S26A/L3H98A, hu6G4.2.5LV/L1S26A,N35A/L3H98A arecollectively referred to herein as “hu6G4.2.5LV/vL1-3A”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV are collectively referred to hereinas “hu6G4.2.5HV”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃, are collectively referredto herein as “hu6G4.2.5HV/H1S31Z₃₁”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A are collectively referred toherein as “hu6G4.2.5HV/H1S31A”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄ are collectively referredto herein as “hu6G4.2.5HV/H2S54Z₅₄”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A are collectively referred toherein as “hu6G4.2.5HV/H2S54A”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3D100E are collectively referredto herein as “hu6G4.2.5HV/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3R102K are collectively referredto herein as “hu6G4.2.5HV/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3D106E are collectively referredto herein as “hu6G4.2.5HV/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3D100E,R102K are collectivelyreferred to herein as “hu6G4.2.5HV/H3D100E,R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3R102K,D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3D100E,D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H3D100E,R102K,D106E arecollectively referred to herein as “hu6G4.2.5HV/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄ are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3D100E are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3R102K are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K arecollectively referred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3R102K,D106E arecollectively referred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3D100E,D106E arecollectively referred to herein as “hu6G4.2.5HV/H1S31Z₃₁/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3D100E are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54Z₅₄/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3R102K are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54Z₅₄/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54Z₅₄/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3R102K,D106E arecollectively referred to herein as “hu6G4.2.5HV/H2S54Z₅₄/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3D100E,D106E arecollectively referred to herein as “hu6G4.2.5HV/H2S54Z₅₄/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54Z₅₄/H3D100E,R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H2S54Z₅₄/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K,D106E are collectivelyreferred to herein as“hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31A/H2S54A”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3D100E are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31A/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3R102K are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31A/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H1S31A/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3D100E,R102K arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H3D100E,R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3R102K,D106E arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3D100E,D106E arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H3D100E,R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31A/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3D100E are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54A/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3R102K are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54A/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3D106E are collectivelyreferred to herein as “hu6G4.2.5HV/H2S54A/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3R102K,D106E arecollectively referred to herein as “hu6G4.2.5HV/H2S54A/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3D100E,D106E arecollectively referred to herein as “hu6G4.2.5HV/H2S54A/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H2S54A/H3D100E,R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H2S54A/H3D100E,R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3D100E arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H2S54A/H3D100E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3R102K arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H2S54A/H3R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3D106E arecollectively referred to herein as “hu6G4.2.5HV/H1S31A/H2S54A/H3D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K arecollectively referred to herein as“hu6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3R102K,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31A/H2S54A/H3R102K,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3D100E,D106E arecollectively referred to herein as“hu6G4.2.5HV/H1S31A/H2S54A/H3D100E,D106E”.

Any and all humanized heavy chain variable domain amino acid sequenceswhich comprise the CDRs of 6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K,D106Eare collectively referred to herein as“hu6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K,D106E”.

The humanized heavy chain variable domain amino acid sequences ofhu6G4.2.5HV/H1S31Z₃₁, hu6G4.2.5HV/H2S54Z₅₄, hu6G4.2.5HV/H3D100E,hu6G4.2.5HV/H3R102K, hu6G4.2.5HV/H3D106E, hu6G4.2.5HV/H3D100E,R102K,hu6G4.2.5HV/H3R102K,D106E, hu6G4.2.5HV/H3D100E,D106E,hu6G4.2.5HV/H3D100E,R102K,D106E, hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄,hu6G4.2.5HV/H1S31Z₃₁/H3D100E, hu6G4.2.5HV/H1S31Z₃₁/H3R102K,hu6G4.2.5HV/H1S31Z₃₁/H3D106E, hu6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K,hu6G4.2.5HV/H1S31Z₃₁/H3R102K,D106E, hu6G4.2.5HV/H1S31Z₃₁/H3D100E,D106E,hu6G4.2.5HV/H1S31Z₃₁/H3D100E,R102K,D106E, hu6G4.2.5HV/H2S54Z₅₄/H3D100E,hu6G4.2.5HV/H2S54Z₅₄/H3R102K, hu6G4.2.5HV/H2S54Z₅₄/H3D106E,hu6G4.2.5HV/H2S54Z₅₄/H3R102K,D106E, hu6G4.2.5HV/H2S54Z₅₄/H3D100E,D106E,hu6G4.2.5HV/H2S54Z₅₄/H3D100E,R102K,D106E,hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K,hu6G4.2.5HV/H1S31Z₃/H2S54Z₅₄/H3D106E,hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K,hu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3R102K,D106E,hu6G4.2.5HV/H1S31Z₃/H2S54Z₅₄/H3D100E,D106E, andhu6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄/H3D100E,R102K,D106E are collectivelyreferred to herein as “hu6G4.2.5HV/vH1-3Z”.

The humanized heavy chain variable domain amino acid sequences ofhu6G4.2.5HV/H1S31A, hu6G4.2.5HV/H2S54A, hu6G4.2.5HV/H3D100E,hu6G4.2.5HV/H3R102K, hu6G4.2.5HV/H3D106E, hu6G4.2.5HV/H3D100E,R102K,hu6G4.2.5HV/H3R102K,D106E, hu6G4.2.5HV/H3D100E,D106E,hu6G4.2.5HV/H3D100E,R102K,D106E, hu6G4.2.5HV/H1S31A/H2S54A,hu6G4.2.5HV/H1S31A/H3D100E, hu6G4.2.5HV/H1S31A/H3R102K,hu6G4.2.5HV/H1S31A/H3D106E, hu6G4.2.5HV/H1S31A/H3D100E,R102K,hu6G4.2.5HV/H1S31A/H3R102K,D106E, hu6G4.2.5HV/H1S31A/H3D100E,D106E,hu6G4.2.5HV/H1S31A/H3D100E,R102K,D106E, hu6G4.2.5HV/H2S54A/H3D100E,hu6G4.2.5HV/H2S54A/H3R102K, hu6G4.2.5HV/H2S54A/H3D106E,hu6G4.2.5HV/H2S54A/H3R102K,D106E, hu6G4.2.5HV/H2S54A/H3D100E,D106E,hu6G4.2.5HV/H2S54A/H3D100E,R102K,D106E,hu6G4.2.5HV/H1S31A/H2S54A/H3D100E, hu6G4.2.5HV/H1S31A/H2S54A/H3R102K,hu6G4.2.5HV/H1S31A/H2S54A/H3D106E,hu6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K,hu6G4.2.5HV/H1S31A/H2S54A/H3R102K,D106E,hu6G4.2.5HV/H1S31A/H2S54A/H3D100E,D106E, andhu6G4.2.5HV/H1S31A/H2S54A/H3D100E,R102K,D106E are collectively referredto herein as “hu6G4.2.5HV/vH1-3A”.

The invention provides a humanized antibody or antibody fragment thatcomprises a light chain variable domain comprising thehu6G4.2.5LV/vL1-3X. In another embodiment, the invention provides ahumanized antibody or antibody fragment that comprises a light chainvariable domain comprising the hu6G4.2.5LV/vL1-3A. In yet anotherembodiment, the invention provides a humanized antibody or antibodyfragment that comprises a light chain variable domain comprising thehu6G4.2.5LV/L1N35X₃₅. In still another embodiment, the inventionprovides a humanized antibody or antibody fragment that comprises alight chain variable domain comprising the hu6G4.2.5LV/L1N35A. In afurther embodiment, the invention provides a humanized antibody orantibody fragment that comprises a light chain variable domaincomprising the hu6G4.2.5LV/L1N35E.

The invention additionally provides a humanized antibody or antibodyfragment that comprises a light chain variable domain comprising thehu6G4.2.5LV/vL1-3X, and further comprises a heavy chain variable domaincomprising the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z. In another embodiment,the invention provides a humanized antibody or antibody fragment thatcomprises a light chain variable domain comprising thehu6G4.2.5LV/vL1-3A, and further comprises a heavy chain variable domaincomprising the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z. In yet anotherembodiment, the invention provides a humanized antibody or antibodyfragment that comprises a light chain variable domain comprising thehu6G4.2.5LV/vL1-3A, and further comprises a heavy chain variable domaincomprising the hu6G4.2.5HV/vH1-3A.

In a further embodiment, the invention provides a humanized antibody orantibody fragment that comprises a light chain variable domaincomprising the hu6G4.2.5LV/L1N35X₃₅, and further comprises a heavy chainvariable domain comprising the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z. Inanother embodiment, the invention provides a humanized antibody orantibody fragment that comprises a light chain variable domaincomprising the hu6G4.2.5LV/N35X₃₅, and further comprises a heavy chainvariable domain comprising the hu6G4.2.5HV/vH1-3A. In a preferredembodiment, the antibody or antibody fragment comprises a light chainvariable domain comprising the hu6G4.2.5LV/L1N35X₃₅ and furthercomprises a humanized heavy chain comprising the amino acid sequence of6G4.2.5HV11.

In an additional embodiment, the invention provides a humanized antibodyor antibody fragment that comprises a light chain variable domaincomprising the hu6G4.2.5LV/L1N35A, and further comprises a heavy chainvariable domain comprising the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z. Inanother embodiment, the invention provides a humanized antibody orantibody fragment that comprises a light chain variable domaincomprising the hu6G4.2.5LV/N35A, and further comprises a heavy chainvariable domain comprising the hu6G4.2.5HV/vH1-3A. In still anotherembodiment, the humanized antibody or antibody fragment comprises alight chain variable domain comprising the hu6G4.2.5LV/L1N35A, andfurther comprises a heavy chain variable domain comprising thehu6G4.2.5HV. In a further embodiment, the humanized antibody or antibodyfragment comprises a light chain variable domain comprising thehu6G4.2.5LV/L1N35E, and further comprises a heavy chain variable domaincomprising the hu6G4.2.5HV. In a preferred embodiment, the antibody orantibody fragment comprises a light chain variable domain comprising thehu6G4.2.5LV/L1N35A and further comprises a humanized heavy chaincomprising the amino acid sequence of 6G4.2.5HV11. In another preferredembodiment, the antibody or antibody fragment comprises a light chainvariable domain comprising the hu6G4.2.5LV/L1N35E and further comprisesa humanized heavy chain comprising the amino acid sequence of6G4.2.5HV11.

The invention encompasses a single chain antibody fragment comprisingthe hu6G4.2.5LV/vL1-3X, with or without any additional amino acidsequence. In one embodiment, the invention provides a single chainantibody fragment comprising the hu6G4.2.5LV/vL1-3X without anyassociated heavy chain variable domain amino acid sequence, i.e. asingle chain species that makes up one half of an Fv fragment. Inanother embodiment, the invention provides a single chain antibodyfragment comprising the hu6G4.2.5LV/vL1-3A without any associated heavychain variable domain amino acid sequence. In still another embodiment,the invention provides a single chain antibody fragment comprising thehu6G4.2.5LV/L1N35X₃₅ without any associated heavy chain variable domainamino acid sequence. In a preferred embodiment, the invention provides asingle chain antibody fragment comprising the hu6G4.2.5LV/L1N35A withoutany associated heavy chain variable domain amino acid sequence. Inanother preferred embodiment, the invention provides a single chainantibody fragment comprising the hu6G4.2.5LV/L1N35E without anyassociated heavy chain variable domain amino acid sequence.

In one embodiment, the invention provides a single chain antibodyfragment wherein the hu6G4.2.5LV/vL1-3X and the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5LV/vL1-3X joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5LV/vL1-3X joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

In another embodiment, the invention provides a single chain antibodyfragment wherein the hu6G4.2.5LV/vL1-3A and the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5LV/vL1-3A joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5LV/vL1-3A joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

In yet another embodiment, the invention provides a single chainantibody fragment wherein the hu6G4.2.5LV/vL1-3A and thehu6G4.2.5HV/vH1-3A are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5LV/vL1-3A joined to thehu6G4.2.5HV/vH1-3A by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5LV/vL1-3A joined to thehu6G4.2.5HV/vH1-3A by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

In still another embodiment, the invention provides a single chainantibody fragment wherein the hu6G4.2.5LV/L1N35X₃₅ and the hu6G4.2.5HVor hu6G4.2.5HV/vH1-3Z are contained in a single chain polypeptidespecies. In a preferred embodiment, the single chain antibody fragmentis a scFv species comprising the hu6G4.2.5LV/L1N35X₃₅ joined to thehu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z by means of a flexible peptide linkersequence, wherein the heavy chain and light chain variable domains canassociate in a “dimeric” structure analogous to that formed in atwo-chain Fv species. In another embodiment, the single chain antibodyfragment is a species comprising the hu6G4.2.5LV/L1N35X₃₅ joined to thehu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z by a linker that is too short topermit intramolecular pairing of the two variable domains, i.e. a singlechain polypeptide monomer that forms a diabody upon dimerization withanother monomer.

In a further embodiment, the invention provides a single chain antibodyfragment wherein the hu6G4.2.5LV/L1N35X₃₅ and the hu6G4.2.5HV/vH1-3A arecontained in a single chain polypeptide species. In a preferredembodiment, the single chain antibody fragment is a scFv speciescomprising the hu6G4.2.5LV/L1N35X₃₅ joined to the hu6G4.2.5HV/vH1-3A bymeans of a flexible peptide linker sequence, wherein the heavy chain andlight chain variable domains can associate in a “dimeric” structureanalogous to that formed in a two-chain Fv species. In anotherembodiment, the single chain antibody fragment is a species comprisingthe hu6G4.2.5LV/L1N35X₃₅ joined to the hu6G4.2.5HV/vH1-3A by a linkerthat is too short to permit intramolecular pairing of the two variabledomains, i.e. a single chain polypeptide monomer that forms a diabodyupon dimerization with another monomer.

In an additional embodiment, the invention provides a single chainantibody fragment wherein the hu6G4.2.5LV/L1N35A and the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5LV/L1N35A joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5LV/L1N35A joined to the hu6G4.2.5HV orhu6G4.2.5HV/vH1-3Z by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

Also provided herein is a single chain antibody fragment wherein thehu6G4.2.5LV/L1N35E and the hu6G4.2.5HV are contained in a single chainpolypeptide species. In a preferred embodiment, the single chainantibody fragment is a scFv species comprising the hu6G4.2.5LV/L1N35Ejoined to the hu6G4.2.5HV by means of a flexible peptide linkersequence, wherein the heavy chain and light chain variable domains canassociate in a “dimeric” structure analogous to that formed in atwo-chain Fv species. In another embodiment, the single chain antibodyfragment is a species comprising the hu6G4.2.5LV/L1N35E joined to thehu6G4.2.5HV by a linker that is too short to permit intramolecularpairing of the two variable domains, i.e. a single chain polypeptidemonomer that forms a diabody upon dimerization with another monomer.

In still another embodiment, the invention provides a single chainantibody fragment wherein the hu6G4.2.5LV/L1N35A and thehu6G4.2.5HV/vH1-3A are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5LV/L1N35A joined to thehu6G4.2.5HV/vH1-3A by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5LV/L1N35A joined to thehu6G4.2.5HV/vH1-3A by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

In yet another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3X and a second polypeptide chaincomprises the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.

In still another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3X and a second polypeptide chaincomprises the hu6G4.2.5HV/vH1-3A and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds. In apreferred embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3X and a second polypeptide chaincomprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.

In a further embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3A and a second polypeptide chaincomprises the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.

In still another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3A and a second polypeptide chaincomprises the hu6G4.2.5HV/vH1-3A and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds. In apreferred embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/vL1-3A and a second polypeptide chaincomprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.

The invention also encompasses an antibody fragment comprising aplurality of polypeptide chains, wherein one polypeptide chain comprisesthe hu6G4.2.5LV/L1N35X₃₅ and a second polypeptide chain comprises thehu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds.

In still another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/L1N35X₃₅ and a second polypeptide chaincomprises the hu6G4.2.5HV/vH1-3A and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds.

In a preferred embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/L1N35X₃₅ and a second polypeptide chaincomprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.

The invention further encompasses an antibody fragment comprising aplurality of polypeptide chains, wherein one polypeptide chain comprisesthe hu6G4.2.5LV/L1N35A and a second polypeptide chain comprises thehu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds.

The invention also encompasses an antibody fragment comprising aplurality of polypeptide chains, wherein one polypeptide chain comprisesthe hu6G4.2.5LV/L1N35E and a second polypeptide chain comprises thehu6G4.2.5HV and the two polypeptide chains are covalently linked by oneor more interchain disulfide bonds.

In still another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/L1N35A and a second polypeptide chaincomprises the hu6G4.2.5HV/vH1-3A and the two polypeptide chains arecovalently linked by one or more interchain disulfide bonds.

In a preferred embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises the hu6G4.2.5LV/L1N35A and a second polypeptide chaincomprises the amino acid sequence of 6G4.2.5HV11 and the two polypeptidechains are covalently linked by one or more interchain disulfide bonds.In another preferred embodiment, the invention provides an antibodyfragment comprising a plurality of polypeptide chains, wherein onepolypeptide chain comprises the hu6G4.2.5LV/L1N35E and a secondpolypeptide chain comprises the amino acid sequence of 6G4.2.5HV11 andthe two polypeptide chains are covalently linked by one or moreinterchain disulfide bonds.

In a preferred embodiment, any of the foregoing two-chain antibodyfragments are selected from the group consisting of Fab, Fab′, Fab′-SH,Fv, and F(ab′)₂. In another preferred embodiment, the antibody fragmentis selected from the group consisting of Fab, Fab′, Fab′-SH, Fv, andF(ab′)₂, wherein the antibody fragment comprises one polypeptide chaincomprising the hu6G4.2.5LV/L1N35X₃₅ and a second polypeptide chaincomprising the hu6G4.2.5HV. In yet another preferred embodiment, theantibody fragment is selected from the group consisting of Fab, Fab′,Fab′-SH, Fv, and F(ab′)₂, wherein the antibody fragment comprises onepolypeptide chain comprising the hu6G4.2.5LV/L1N35A and a secondpolypeptide chain comprising the hu6G4.2.5HV. In a further preferredembodiment, the antibody fragment is selected from the group consistingof Fab, Fab′, Fab′-SH, Fv, and F(ab′)₂, wherein the antibody fragmentcomprises one polypeptide chain comprising the hu6G4.2.5LV/L1N35E and asecond polypeptide chain comprising the hu6G4.2.5HV. In still anotherpreferred embodiment, the antibody fragment is a F(ab′)₂ that comprisesone polypeptide chain comprising the hu6G4.2.5LV/L1N35A and a secondpolypeptide chain comprising the amino acid sequence of 6G4.2.5HV11. Inan additional preferred embodiment, the antibody fragment is a F(ab′)₂that comprises one polypeptide chain comprising the hu6G4.2.5LV/L1N35Eand a second polypeptide chain comprising the amino acid sequence of6G4.2.5HV11.

The invention also provides an antibody or antibody fragment comprisinga light chain variable domain containing the hu6G4.2.5LV/vL1-3X andoptionally further comprising a heavy chain variable domain containingthe hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z, wherein the light chain variabledomain, and optionally the heavy chain variable domain, is (are) fusedto an additional moiety, such as a immunoglobulin constant domain.Constant domain sequence can be added to the heavy chain and/or lightchain sequence(s) to form species with full or partial length heavyand/or light chain(s). It will be appreciated that constant regions ofany isotype can be used for this purpose, including IgG, IgM, IgA, IgD,and IgE constant regions, and that such constant regions can be obtainedfrom any human or animal species. Preferably, the constant domainsequence is human in origin. Suitable human constant domain sequencescan be obtained from Kabat et al.

The invention additionally provides an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/vL1-3X and optionally further comprising a heavy chainvariable domain containing the hu6G4.2.5HV/vH1-3A, wherein the lightchain variable domain, and optionally the heavy chain variable domain,is (are) fused to an additional moiety, such as a immunoglobulinconstant domain. Constant domain sequence can be added to the heavychain and/or light chain sequence(s) to form species with full orpartial length heavy and/or light chain(s). It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species. Preferably,the constant domain sequence is human in origin. Suitable human constantdomain sequences can be obtained from Kabat et al.

The invention further provides an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35X₃₅ and optionally further comprising a heavy chainvariable domain containing the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z,wherein the light chain variable domain, and optionally the heavy chainvariable domain, is (are) fused to an additional moiety, such as aimmunoglobulin constant domain. Constant domain sequence can be added tothe heavy chain and/or light chain sequence(s) to form species with fullor partial length heavy and/or light chain(s). It will be appreciatedthat constant regions of any isotype can be used for this purpose,including IgG, IgM, IgA, IgD, and IgE constant regions, and that suchconstant regions can be obtained from any human or animal species.Preferably, the constant domain sequence is human in origin. Suitablehuman constant domain sequences can be obtained from Kabat et al.

The invention additionally provides an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35X₃₅ and optionally further comprising a heavy chainvariable domain containing the hu6G4.2.5HV/vH1-3A, wherein the lightchain variable domain, and optionally the heavy chain variable domain,is (are) fused to an additional moiety, such as a immunoglobulinconstant domain. Constant domain sequence can be added to the heavychain and/or light chain sequence(s) to form species with full orpartial length heavy and/or light chain(s). It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species. Preferably,the constant domain sequence is human in origin. Suitable human constantdomain sequences can be obtained from Kabat et al.

The invention also encompasses an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35A and optionally further comprising a heavy chainvariable domain containing the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z,wherein the light chain variable domain, and optionally the heavy chainvariable domain, is (are) fused to an additional moiety, such as aimmunoglobulin constant domain. Constant domain sequence can be added tothe heavy chain and/or light chain sequence(s) to form species with fullor partial length heavy and/or light chain(s). It will be appreciatedthat constant regions of any isotype can be used for this purpose,including IgG, IgM, IgA, IgD, and IgE constant regions, and that suchconstant regions can be obtained from any human or animal species.Preferably, the constant domain sequence is human in origin. Suitablehuman constant domain sequences can be obtained from Kabat et al.

The invention additionally provides an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35A and optionally further comprising a heavy chainvariable domain containing the hu6G4.2.5HV/vH1-3A, wherein the lightchain variable domain, and optionally the heavy chain variable domain,is (are) fused to an additional moiety, such as a immunoglobulinconstant domain. Constant domain sequence can be added to the heavychain and/or light chain sequence(s) to form species with full orpartial length heavy and/or light chain(s). It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species. Preferably,the constant domain sequence is human in origin. Suitable human constantdomain sequences can be obtained from Kabat et al.

The invention additionally encompasses an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35A and optionally further comprising a heavy chaincontaining the amino acid sequence of 6G4.2.5HV111, wherein the lightchain variable domain, and optionally the heavy chain, is (are) fused toan additional moiety, such as immunoglobulin constant domain sequences.Constant domain sequence can be added to the heavy chain and/or lightchain sequence(s) to form species with full or partial length heavyand/or light chain(s). It will be appreciated that constant regions ofany isotype can be used for this purpose, including IgG, IgM, IgA, IgD,and IgE constant regions, and that such constant regions can be obtainedfrom any human or animal species. Preferably, the constant domainsequence is human in origin. Suitable human constant domain sequencescan be obtained from Kabat et al.

The invention further encompasses an antibody or antibody fragmentcomprising a light chain variable domain containing thehu6G4.2.5LV/L1N35E and optionally further comprising a heavy chaincontaining the amino acid sequence of 6G4.2.5HV11, wherein the lightchain variable domain, and optionally the heavy chain, is (are) fused toan additional moiety, such as immunoglobulin constant domain sequences.Constant domain sequence can be added to the heavy chain and/or lightchain sequence(s) to form species with full or partial length heavyand/or light chain(s). It will be appreciated that constant regions ofany isotype can be used for this purpose, including IgG, IgM, IgA, IgD,and IgE constant regions, and that such constant regions can be obtainedfrom any human or animal species. Preferably, the constant domainsequence is human in origin. Suitable human constant domain sequencescan be obtained from Kabat et al.

In a preferred embodiment, the antibody or antibody fragment comprises alight chain variable domain containing the hu6G4.2.5LV/vL1-3X, andfurther comprises the hu6G4.2.5HV or hu6G4.2.5HV/vH1-3Z in a heavy chainthat is fused to or contains a leucine zipper sequence. The leucinezipper can increase the affinity or production efficiency of theantibody or antibody fragment of interest. Suitable leucine zippersequences include the jun and fos leucine zippers taught by Kostelney etal., J. Immunol., 148: 1547–1553 (1992) and the GCN4 leucine zipperdescribed in the Examples below.

In particular, the invention provides an antibody or antibody fragmentcomprising a light chain comprising the amino acid sequence of aminoacids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) with theproviso that any amino acid other than Asn (denoted as “X₃₅”) issubstituted for Asn at amino acid position 35 (herein referred to as“6G4.2.5LV11N35X₃₅”).

In another embodiment, the invention provides an antibody or antibodyfragment comprising a light chain comprising the amino acid sequence ofamino acids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 lightchain polypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) withthe proviso that any amino acid other than Ser (denoted as “X₂₆”) issubstituted for Ser at amino acid position 26 (herein referred to as“6G4.2.5LV11S26X₂₆”).

In yet another embodiment, the invention provides an antibody orantibody fragment comprising a light chain comprising the amino acidsequence of amino acids 1–219 of the variant humanized anti-IL-86G4.2.5v11 light chain polypeptide amino acid sequence of FIG. 31B (SEQID NO: 51) with the proviso that any amino acid other than His (denotedas “X₉₈”) is substituted for His at amino acid position 98 (hereinreferred to as “6G4.2.5LV11H98X₉₈”).

In still another embodiment, the invention provides an antibody orantibody fragment comprising a light chain comprising the amino acidsequence of amino acids 1–219 of the variant humanized anti-IL-86G4.2.5v11 light chain polypeptide amino acid sequence of FIG. 31B (SEQID NO: 51) with the proviso that any amino acid other than Ser (denotedas “X₂₆”) is substituted for Ser at amino acid position 26 and any aminoacid other than Asn (denoted as “X₃₅”) is substituted for Asn at aminoacid position 35 (herein referred to as “6G4.2.5LV11S26X₂₆/N35X₃₅”).

In a further embodiment, the invention provides an antibody or antibodyfragment comprising a light chain comprising the amino acid sequence ofamino acids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 lightchain polypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) withthe proviso that any amino acid other than Asn (denoted as “X₃₅”) issubstituted for Asn at amino acid position 35 and any amino acid otherthan His (denoted as “X₉₈”) is substituted for His at amino acidposition 98 (herein referred to as “6G4.2.5LV11N35X₃₅/H98X₉₈”).

In an additional embodiment, the invention provides an antibody orantibody fragment comprising a light chain comprising the amino acidsequence of amino acids 1–219 of the variant humanized anti-IL-86G4.2.5v11 light chain polypeptide amino acid sequence of FIG. 31B (SEQID NO: 51) with the proviso that any amino acid other than Ser (denotedas “X₂₆”) is substituted for Ser at amino acid position 26 and any aminoacid other than His (denoted as “X₉₈”) is substituted for His at aminoacid position 98 (herein referred to as “6G4.2.5LV11S26X₂₆/H98X₉₈”).

The invention also encompasses an antibody or antibody fragmentcomprising a light chain comprising the amino acid sequence of aminoacids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) with theproviso that any amino acid other than Ser (denoted as “X₂₆”) issubstituted for Ser at amino acid position 26, any amino acid other thanAsn (denoted as “X₃₅”) is substituted for Asn at amino acid position 35and any amino acid other than His (denoted as “X₉₈”) is substituted forHis at amino acid position 98 (herein referred to as“6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈”).

Additionally, the invention provides an antibody or antibody fragmentcomprising a light chain comprising the amino acid sequence of aminoacids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence (SEQ ID NO: 56) of FIG. 36 (hereinreferred to as “6G4.2.5LV11N35A”).

Further provided herein is an antibody or antibody fragment comprising alight chain comprising the amino acid sequence of amino acids 1–219 ofthe variant humanized anti-IL-8 6G4.2.5v11 light chain polypeptide aminoacid sequence (SEQ ID NO: 62) of FIG. 45 (herein referred to as“6G4.2.5LV11N35E”).

In another embodiment, the invention provides an antibody or antibodyfragment comprising a light chain comprising the amino acid sequence ofamino acids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 lightchain polypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) withthe proviso that Ala is substituted for Ser at amino acid position 26(herein referred to as “6G4.2.5LV11S26A”).

In yet another embodiment, the invention provides an antibody orantibody fragment comprising a light chain comprising the amino acidsequence of amino acids 1–219 of the variant humanized anti-IL-86G4.2.5v11 light chain polypeptide amino acid sequence of FIG. 31B (SEQID NO: 51) with the proviso that Ala is substituted for His at aminoacid position 98 (herein referred to as “6G4.2.5LV11H98A”).

In still another embodiment, the invention provides an antibody orantibody fragment comprising a light chain comprising the amino acidsequence of amino acids 1–219 of the variant humanized anti-IL-86G4.2.5v11 light chain polypeptide amino acid sequence of FIG. 31B (SEQID NO: 51) with the proviso that Ala is substituted for Ser at aminoacid position 26 and Ala is substituted for Asn at amino acid position35 (herein referred to as “6G4.2.5LV11S26A/N35A”).

In a further embodiment, the invention provides an antibody or antibodyfragment comprising a light chain comprising the amino acid sequence ofamino acids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 lightchain polypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) withthe proviso that Ala is substituted for Ser at amino acid position 26and Ala is substituted for His at amino acid position 98 (hereinreferred to as “6G4.2.5LV11S26A/H98A”).

The invention also encompasses an antibody or antibody fragmentcomprising a light chain comprising the amino acid sequence of aminoacids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) with theproviso that Ala is substituted for Asn at amino acid position 35 andAla is substituted for His at amino acid position 98 (herein referred toas “6G4.2.5LV11N35A/H98A”).

The invention further encompasses an antibody or antibody fragmentcomprising a light chain comprising the amino acid sequence of aminoacids 1–219 of the variant humanized anti-IL-8 6G4.2.5v11 light chainpolypeptide amino acid sequence of FIG. 31B (SEQ ID NO: 51) with theproviso that Ala is substituted for Ser at amino acid position 26, Alais substituted for Asn at amino acid position 35, and Ala is substitutedfor His at amino acid position 98 (herein referred to as“6G4.2.5LV11S26A/N35A/H98A”).

The invention provides a single chain antibody fragment comprising avariant light chain selected from the group consisting of6G4.2.5LV11N35X₃₅, 6G4.2.5LV11S26X₂₆, 6G4.2.5LV11H98X₉₈,6G4.2.5LV11S26X₂₆/N35X₃₅, 6G4.2.5LV11N35X₃₅/H98X₉₈,6G4.2.5LV11S26X₂₆/H98X₈, and 6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈, with orwithout any additional amino acid sequence. It will be understood thatthe group consisting of 6G4.2.5LV11N35X₃₅, 6G4.2.5LV11S26X₂₆,6G4.2.5LV11H98X₉₈, 6G4.2.5LV11S26X₂₆/N35X₃₅, 6G4.2.5LV11N35X₃₅/H98X₉₈,6G4.2.5LV11S26X₂₆/H98X₉₈, and 6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈, iscollectively referred to herein as the “group of 6G4.2.5LV11X variants”,and that individual members of this group are generically referred toherein as a “6G4.2.5LV11X variant.” In one embodiment, the inventionprovides a single chain antibody fragment comprising a 6G4.2.5LV11Xvariant without any associated heavy chain amino acid sequence, i.e. asingle chain species that makes up one half of a Fab fragment. In apreferred embodiment, the invention provides a 6G4.2.5LV11N35X₃₅ variantwithout any associated heavy chain amino acid sequence.

The invention encompasses a single chain antibody fragment comprising avariant light chain selected from the group consisting of6G4.2.5LV11N35A, 6G4.2.5LV11S26A. 6G4.2.5LV11H98A, 6G4.2.5LV11S26A/N35A,6G4.2.5LV11N35A/H98A, 6G4.2.5LV11S26A/H98A, and6G4.2.5LV11S26A/N35A/H98A, with or without any additional amino acidsequence. It will be understood that the group consisting of6G4.2.5LV11N35A, 6G4.2.5LV11S26A, 6G4.2.5LV11H98A, 6G4.2.5LV11S26A/N35A,6G4.2.5LV11N35A/H98A, 6G4.2.5LV11S26A/H98A, and6G4.2.5LV11S26A/N35A/H98A is collectively referred to herein as the“group of 6G4.2.5LV11A variants”, and that individual members of thisgroup are generically referred to herein as a “6G4.2.5LV11A variant.” Inone embodiment, the invention provides a single chain antibody fragmentcomprising a 6G4.2.5LV11A variant without any associated heavy chainamino acid sequence, i.e. a single chain species that makes up one halfof a Fab fragment. In a preferred embodiment, the invention provides the6G4.2.5LV11N35A without any associated heavy chain amino acid sequence.

Further provided herein are an antibody or antibody fragment comprisinga light chain comprising a 6G4.2.5LV11X variant, and further comprisinga heavy chain comprising the 6G4.2.5HV11. In a preferred embodiment, theinvention provides an antibody or antibody fragment comprising a6G4.2.5LV11N35X₃₅ variant and further comprising the 6G4.2.5HV11. In apreferred embodiment, the invention provides an antibody or antibodyfragment comprising the 6G4.2.5LV11N35A and further comprising the6G4.2.5HV11. In another preferred embodiment, the invention provides anantibody or antibody fragment comprising the 6G4.2.5LV11N35E and furthercomprising the 6G4.2.5HV11.

In one embodiment, the invention provides a single chain antibodyfragment wherein a 6G4.2.5LV11X variant and the 6G4.2.5HV11 arecontained in a single chain polypeptide species. In a preferredembodiment, the single chain antibody fragment comprises a 6G4.2.5LV11Xvariant joined to the 6G4.2.5HV11 by means of a flexible peptide linkersequence, wherein the heavy chain and light chain domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fabspecies. In another embodiment, the single chain antibody fragment is aspecies comprising a 6G4.2.5LV11X variant joined to the 6G4.2.5HV11 by alinker that is too short to permit intramolecular pairing ofcomplementary domains, i.e. a single chain polypeptide monomer thatforms a diabody upon dimerization with another monomer.

In still another embodiment, the invention provides a single chainantibody fragment wherein a 6G4.2.5LV11N35X₃₅ variant and the6G4.2.5HV11 are contained in a single chain polypeptide species. In apreferred embodiment, the single chain antibody fragment comprises a6G4.2.5LV11N35X₃₅ variant joined to the 6G4.2.5HV11 by means of aflexible peptide linker sequence, wherein the heavy chain and lightchain domains can associate in a “‘dimeric’” structure analogous to thatformed in a two-chain Fab species. In another embodiment, the singlechain antibody fragment is a species comprising a 6G4.2.5LV11N35X₃₅variant joined to the 6G4.2.5HV11 by a linker that is too short topermit intramolecular pairing of complementary domains, i.e. a singlechain polypeptide monomer that forms a diabody upon dimerization withanother monomer.

In a further embodiment, the invention provides a single chain antibodyfragment wherein the 6G4.2.5LV11N35A and the 6G4.2.5HV11 are containedin a single chain polypeptide species. In a preferred embodiment, thesingle chain antibody fragment comprises the 6G4.2.5LV11N35A joined tothe 6G4.2.5HV11 by means of a flexible peptide linker sequence, whereinthe heavy chain and light chain domains can associate in a “dimeric”structure analogous to that formed in a two-chain Fab species. Inanother embodiment, the single chain antibody fragment is a speciescomprising the 6G4.2.5LV11N35A joined to the 6G4.2.5HV11 by a linkerthat is too short to permit intramolecular pairing of complementarydomains, i.e. a single chain polypeptide monomer that forms a diabodyupon dimerization with another monomer.

In an additional embodiment, the invention provides a single chainantibody fragment wherein the 6G4.2.5LV11N35E and the 6G4.2.5HV11 arecontained in a single chain polypeptide species. In a preferredembodiment, the single chain antibody fragment comprises the6G4.2.5LV11N35E joined to the 6G4.2.5HV11 by means of a flexible peptidelinker sequence, wherein the heavy chain and light chain domains canassociate in a “dimeric” structure analogous to that formed in atwo-chain Fab species. In another embodiment, the single chain antibodyfragment is a species comprising the 6G4.2.5LV11N35E joined to the6G4.2.5HV11 by a linker that is too short to permit intramolecularpairing of complementary domains, i.e. a single chain polypeptidemonomer that forms a diabody upon dimerization with another monomer.

In yet another embodiment, the invention provides an antibody fragmentcomprising a plurality of polypeptide chains, wherein one polypeptidechain comprises a 6G4.2.5LV11X variant and a second polypeptide chaincomprises the 6G4.2.5HV11 and the two polypeptide chains are covalentlylinked by one or more interchain disulfide bonds. In still anotherembodiment, the invention provides an antibody fragment comprising aplurality of polypeptide chains, wherein one polypeptide chain comprisesa 6G4.2.5LV11N35X₃₅ variant and a second polypeptide chain comprises the6G4.2.5HV11 and the two polypeptide chains are covalently linked by oneor more interchain disulfide bonds. In a preferred embodiment, any ofthe foregoing two-chain antibody fragments is selected from the groupconsisting of Fab, Fab′, Fab′-SH, and F(ab′)₂. In still anotherpreferred embodiment, the two-chain antibody fragment is a F(ab′)₂wherein one polypeptide chain comprises the 6G4.2.5LV11N35A and thesecond polypeptide chain comprises the 6G4.2.5HV11. In a furtherpreferred embodiment, the antibody fragment is a Fab, Fab′, Fab′-SH, orF(ab′)₂ wherein one polypeptide chain comprises the 6G4.2.5LV11N35E andthe second polypeptide chain comprises the 6G4.2.5HV11. A particularlypreferred embodiment, the antibody fragment is the 6G4V11N35A F(ab′)₂GCN4 leucine zipper species described in the Examples below. In anotherparticularly preferred embodiment, the antibody fragment is the6G4V11N35E F(ab′)₂ GCN4 leucine zipper species described in the Examplesbelow. In yet another particularly preferred embodiment, the antibodyfragment is the 6G4V11N35E Fab described in the Examples below.

The invention also provides an antibody or antibody fragment comprisinga light chain containing a 6G4.2.5LV11X variant and optionally furthercomprising a heavy chain containing the 6G4.2.5HV11, wherein the lightchain, and optionally the heavy chain, is (are) fused to an additionalmoiety, such as additional immunoglobulin constant domain sequence.Constant domain sequence can be added to the heavy chain and/or lightchain sequence(s) to form species with full or partial length heavyand/or light chain(s). It will be appreciated that constant regions ofany isotype can be used for this purpose, including IgG, IgM, IgA, IgD,and IgE constant regions, and that such constant regions can be obtainedfrom any human or animal species. Preferably, the constant domainsequence is human in origin. Suitable human constant domain sequencescan be obtained from Kabat et al.

The invention additionally provides an antibody or antibody fragmentcomprising a light chain containing a 6G4.2.5LV11N35X₃₅ variant andoptionally further comprising a heavy chain containing the 6G4.2.5HV11,wherein the light chain, and optionally the heavy chain, is (are) fusedto an additional moiety, such as additional immunoglobulin constantdomain sequence. Constant domain sequence can be added to the heavychain and/or light chain sequence(s) to form species with full orpartial length heavy and/or light chain(s). It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species. Preferably,the constant domain sequence is human in origin. Suitable human constantdomain sequences can be obtained from Kabat et al.

The invention further provides an antibody or antibody fragmentcomprising a light chain containing the 6G4.2.5LV11N35A and optionallyfurther comprising a heavy chain containing the 6G4.2.5HV11, wherein thelight chain, and optionally the heavy chain, is (are) fused to anadditional moiety, such as additional immunoglobulin constant domainsequence. Constant domain sequence can be added to the heavy chainand/or light chain sequence(s) to form species with full or partiallength heavy and/or light chain(s). It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species. Preferably, the constantdomain sequence is human in origin. Suitable human constant domainsequences can be obtained from Kabat et al.

The invention further provides an antibody or antibody fragmentcomprising a light chain containing the 6G4.2.5LV11N35E and optionallyfurther comprising a heavy chain containing the 6G4.2.5HV11, wherein thelight chain, and optionally the heavy chain, is (are) fused to anadditional moiety, such as additional immunoglobulin constant domainsequence. Constant domain sequence can be added to the heavy chainand/or light chain sequence(s) to form species with full or partiallength heavy and/or light chain(s). It will be appreciated that constantregions of any isotype can be used for this purpose, including IgG, IgM,IgA, IgD, and IgE constant regions, and that such constant regions canbe obtained from any human or animal species. Preferably, the constantdomain sequence is human in origin. Suitable human constant domainsequences can be obtained from Kabat et al.

In a preferred embodiment, the antibody or antibody fragment comprises alight chain containing a 6G4.2.5LV11X variant, and further comprises the6G4.2.5HV11 in a heavy chain that is fused to or contains a leucinezipper sequence. The leucine zipper can increase the affinity orproduction efficiency of the antibody or antibody fragment of interest.Suitable leucine zipper sequences include the jun and fos leucinezippers taught by Kostelney et al., J. Immunol., 148: 1547–1553 (1992)and the GCN4 leucine zipper described in the Examples below. In anotherpreferred embodiment, the antibody or antibody fragment comprises alight chain containing the 6G4.2.5LV11N35A, and further comprises aheavy chain containing the 6G4.2.5HV11 fused to the GCN4 leucine zipper.In yet another preferred embodiment, the antibody or antibody fragmentcomprises a light chain containing the 6G4.2.5LV11N35E, and furthercomprises a heavy chain containing the 6G4.2.5HV11 fused to the GCN4leucine zipper.

B. 6G4.2.5HV Variants

The invention provides humanized antibodies and antibody fragmentscomprising the CDRs of a 6G4.2.5HV CDR variant. The use of a 6G4.2.5HVCDRs variant in the humanized antibodies and antibody fragments of theinvention confer the advantages of higher affinity for human IL-8 and/orimproved recombinant manufacturing economy.

A heavy chain variable domain comprising the CDRs of a 6G4.2.5HV CDRsvariant can be humanized in conjunction with a light chain comprisingthe CDRs of 6G4.2.5LV or the CDRs of a 6G4.2.5LV CDRs variant,essentially as described in Section (II)(2)(A) above. In one embodiment,the invention provides a humanized antibody or antibody fragmentcomprising a 6G4.2.5HV CDRs variant selected from the group consistingof 6G4.2.5HV/H1S31Z₃₁, 6G4.2.5HV/H2S54Z₅₄, and6G4.2.5HV/H1S31Z₃₁/H2S54Z₅₄. In addition, the invention provides ahumanized antibody or antibody fragment comprising a 6G4.2.5HV CDRsvariant selected from the group consisting of 6G4.2.5HV/H1S31A,6G4.2.5HV/H2S54A, and 6G4.2.5HV/H1S31A/H2S54A. In particular, the6G4.2.5HV CDRs variants can be used to construct a humanized antibody orantibody comprising the hu6G4.2.5HV/vH1-3Z as described in Section(II)(2)(A) above.

The invention additionally provides a humanized antibody or antibodyfragment that comprises a heavy chain variable domain comprising thehu6G4.2.5HV/vH1-3Z, and further comprises a light chain variable domaincomprising the hu6G4.2.5LV or hu6G4.2.5LV/vL1-3X.

The invention further encompasses a single chain humanized antibodyfragment comprising the hu6G4.2.5HV/vH1-3Z, with or without anyadditional amino acid sequence. In one embodiment, the inventionprovides a single chain antibody fragment comprising thehu6G4.2.5HV/vH1-3Z without any associated heavy chain variable domainamino acid sequence, i.e. a single chain species that makes up one halfof an Fv fragment.

In one embodiment, the invention provides a single chain humanizedantibody fragment wherein the hu6G4.2.5HV/vH1-3Z and the hu6G4.2.5LV orhu6G4.2.5LV/vL1-3X are contained in a single chain polypeptide species.In a preferred embodiment, the single chain antibody fragment is a scFvspecies comprising the hu6G4.2.5HV/vH1-3Z joined to the hu6G4.2.5LV orhu6G4.2.5LV/vL1-3X by means of a flexible peptide linker sequence,wherein the heavy chain and light chain variable domains can associatein a “dimeric” structure analogous to that formed in a two-chain Fvspecies. In another embodiment, the single chain antibody fragment is aspecies comprising the hu6G4.2.5HV/vH1-3Z joined to the hu6G4.2.5LV orhu6G4.2.5LV/vL1-3X by a linker that is too short to permitintramolecular pairing of the two variable domains, i.e. a single chainpolypeptide monomer that forms a diabody upon dimerization with anothermonomer.

In yet another embodiment, the invention provides a humanized antibodyfragment comprising a plurality of polypeptide chains, wherein onepolypeptide chain comprises the hu6G4.2.5HV/vH1-3Z and a secondpolypeptide chain comprises the hu6G4.2.5LV or hu6G4.2.5LV/vL1-3X andthe two polypeptide chains are covalently linked by one or moreinterchain disulfide bonds. In a preferred embodiment, the foregoingtwo-chain antibody fragment is selected from the group consisting ofFab, Fab′, Fab′-SH, Fv, and F(ab′)₂.

The invention also provides a humanized antibody or antibody fragmentcomprising a heavy chain variable domain containing thehu6G4.2.5HV/vH1-3Z and optionally further comprising a light chainvariable domain containing the hu6G4.2.5LV or hu6G4.2.5LV/vL1-3X,wherein the heavy chain variable domain, and optionally the light chainvariable domain, is (are) fused to an additional moiety, such as animmunoglobulin constant domain. Constant domain sequence can be added tothe heavy chain and/or light chain sequence(s) to form species with fullor partial length heavy and/or light chain(s). It will be appreciatedthat constant regions of any isotype can be used for this purpose,including IgG, IgM, IgA, IgD, and IgE constant regions, and that suchconstant regions can be obtained from any human or animal species.Preferably, the constant domain sequence is human in origin. Suitablehuman constant domain sequences can be obtained from Kabat et al.

In a preferred embodiment, the humanized antibody or antibody fragmentcomprises the hu6G4.2.5HV/vH1-3Z in a heavy chain that is fused to orcontains a leucine zipper sequence. The leucine zipper can increase theaffinity or production efficiency of the antibody or antibody fragmentof interest. Suitable leucine zipper sequences include the jun and fosleucine zippers taught by Kostelney et al., J. Immunol., 148: 1547–1553(1992) and the GCN4 leucine zipper described in the Examples below.

In addition, the invention provides a humanized antibody or antibodyfragment comprising a heavy chain comprising the amino acid sequence ofamino acids 1–230 of the 6G4.2.5HV11 polypeptide amino acid sequence ofFIGS. 37A–37B (SEQ ID NO: 60) with the proviso that Ala is substitutedfor Ser at amino acid position 31 (hereinafter referred to as“6G4.2.5HV11S31A”).

In another embodiment, the invention provides a humanized antibody orantibody fragment comprising a heavy chain comprising the amino acidsequence of amino acids 1–230 of the 6G4.2.5HV11 polypeptide amino acidsequence of FIGS. 37A–37B (SEQ ID NO: 60) with the proviso that Ala issubstituted for Ser at amino acid position 54 (hereinafter referred toas “6G4.2.5HV11S54A”).

In yet another embodiment, the invention provides a humanized antibodyor antibody fragment comprising a heavy chain comprising the amino acidsequence of amino acids 1–230 of the 6G4.2.5HV11 polypeptide amino acidsequence of FIGS. 37A–37B (SEQ ID NO: 60) with the proviso that Ala issubstituted for Ser at amino acid position 31 and Ala is substituted forSer at amino acid position 54 (hereinafter referred to as“6G4.2.5HV11S31A/S54A”).

Further provided herein is a humanized antibody or antibody fragmentthat comprises any of the light and heavy chain combinations listed inTables 1–2 below.

TABLE 1 Heavy Chain Light Chain 6G4.2.5HV11S31A 6G4.2.5LV116G4.2.5HV11S31A 6G4.2.5LV11N35A 6G4.2.5HV11S31A 6G4.2.5LV11S26A6G4.2.5HV11S31A 6G4.2.5LV11H98A 6G4.2.5HV11S31A 6G4.2.5LV11S26A/N35A6G4.2.5HV11S31A 6G4.2.5LV11S26A/H98A 6G4.2.5HV11S31A6G4.2.5LV11N35A/H98A 6G4.2.5HV11S31A 6G4.2.5LV11S26A/N35A/H98A6G4.2.5HV11S54A 6G4.2.5LV11 6G4.2.5HV11S54A 6G4.2.5LV11N35A6G4.2.5HV11S54A 6G4.2.5LV11S26A 6G4.2.5HV11S54A 6G4.2.5LV11H98A6G4.2.5HV11S54A 6G4.2.5LV11S26A/N35A 6G4.2.5HV11S54A6G4.2.5LV11S26A/H98A 6G4.2.5HV11S54A 6G4.2.5LV11N35A/H98A6G4.2.5HV11S54A 6G4.2.5LV11S26A/N35A/H98A 6G4.2.5HV11S31A/S54A6G4.2.5LV11 6G4.2.5HV11S31A/S54A 6G4.2.5LV11N35A 6G4.2.5HV11S31A/S54A6G4.2.5LV11S26A 6G4.2.5HV11S31A/S54A 6G4.2.5LV11H98A6G4.2.5HV11S31A/S54A 6G4.2.5LV11S26A/N35A 6G4.2.5HV11S31A/S54A6G4.2.5LV11S26A/H98A 6G4.2.5HV11S31A/S54A 6G4.2.5LV11N35A/H98A6G4.2.5HV11S31A/S54A 6G4.2.5LV11S26A/N35A/H98A

TABLE 2 Heavy Chain Light Chain 6G4.2.5HV11S31A 6G4.2.5LV116G4.2.5HV11S31A 6G4.2.5LV11N35X₃₅ 6G4.2.5HV11S31A 6G4.2.5LV11S26X₂₆6G4.2.5HV11S31A 6G4.2.5LV11H98X₉₈ 6G4.2.5HV11S31A6G4.2.5LV11S26X₂₆/N35X₃₅ 6G4.2.5HV11S31A 6G4.2.5LV11S26X₂₆/H98X₉₈6G4.2.5HV11S31A 6G4.2.5LV11N35X₃₅/H98X₉₈ 6G4.2.5HV11S31A6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈ 6G4.2.5HV11S54A 6G4.2.5LV116G4.2.5HV11S54A 6G4.2.5LV11N35X₃₅ 6G4.2.5HV11S54A 6G4.2.5LV11S26X₂₆6G4.2.5HV11S54A 6G4.2.5LV11H98X₉₈ 6G4.2.5HV11S54A6G4.2.5LV11S26X₂₆/N35X₃₅ 6G4.2.5HV11S54A 6G4.2.5LV11S26X₂₆/H98X₉₈6G4.2.5HV11S54A 6G4.2.5LV11N35X₃₅/H98X₉₈ 6G4.2.5HV11S54A6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈ 6G4.2.5HV11S31A/S54A 6G4.2.5LV116G4.2.5HV11S31A/S54A 6G4.2.5LV11N35X₃₅ 6G4.2.5HV11S31A/S54A6G4.2.5LV11S26X₂₆ 6G4.2.5HV11S31A/S54A 6G4.2.5LV11H98X₉₈6G4.2.5HV11S31A/S54A 6G4.2.5LV11S26X₂₆/N35X₃₅ 6G4.2.5HV11S31A/S54A6G4.2.5LV11S26X₂₆/H98X₉₈ 6G4.2.5HV11S31A/S54A 6G4.2.5LV11N35X₃₅/H98X₉₈6G4.2.5HV11S31A/S54A 6G4.2.5LV11S26X₂₆/N35X₃₅/H98X₉₈

The invention encompasses a single chain humanized antibody fragmentcomprising a variant heavy chain selected from the group consisting of6G4.2.5HV11S31A, 6G4.2.5HV11S54A, and 6G4.2.5HV11S31A/S54A, with orwithout any additional amino acid sequence. It will be understood thatthe group consisting of 6G4.2.5HV11S31A, 6G4.2.5HV11S54A, and6G4.2.5HV11S31A/S54A is collectively referred to herein as the “group of6G4.2.5HV11A variants”, and that individual members of this group aregenerically referred to herein as a “6G4.2.5HV11A variant.” In oneembodiment, the invention provides a single chain humanized antibodyfragment comprising a 6G4.2.5HV11A variant without any associated lightchain amino acid sequence, i.e. a single chain species that makes up onehalf of a Fab fragment.

Further provided herein are a humanized antibody or antibody fragmentcomprising a heavy chain comprising a 6G4.2.5HV11A variant, and furthercomprising a light chain comprising a 6G4.2.5LV11A variant or a6G4.2.5LV11X variant. In another embodiment, the humanized antibody orantibody fragment comprises any combination of light and heavy chainslisted in Tables 1 and 2 above. In one embodiment, the inventionprovides a humanized antibody or antibody fragment comprising a6G4.2.5HV11A variant and further comprising the 6G4.2.5LV11N35X₃₅. In apreferred embodiment, the invention provides a humanized antibody orantibody fragment comprising a 6G4.2.5HV11A variant and furthercomprising the 6G4.2.5LV11N35A.

In yet another embodiment, the invention provides a single chainhumanized antibody fragment wherein a 6G4.2.5HV11A variant and the6G4.2.5LV11 are contained in a single chain polypeptide species. Inanother embodiment, the invention provides a single chain humanizedantibody fragment wherein any pair of light and heavy chains listed inTables 1–2 above is contained in a single chain polypeptide species. Inyet another embodiment, the invention provides a single chain humanizedantibody fragment wherein a 6G4.2.5HV11A variant and a 6G4.2.5LV11Xvariant are contained in a single chain polypeptide species. In stillanother embodiment, the invention provides a single chain humanizedantibody fragment wherein a 6G4.2.5HV11A variant and a 6G4.2.5LV11N35X₃₅variant are contained in a single chain polypeptide species. In anadditional embodiment, the invention provides a single chain humanizedantibody fragment wherein a 6G4.2.5HV11A variant and the 6G4.2.5LV11N35Avariant are contained in a single chain polypeptide species.

In a preferred embodiment, the single chain humanized antibody fragmentcomprises a 6G4.2.5HV11A variant joined to a 6G4.2.5LV11X variant,6G4.2.5LV11N35X₃, variant, 6G4.2.5LV11N35A variant, or 6G4.2.5LV11 bymeans of a flexible peptide linker sequence, wherein the heavy chain andlight chain domains can associate in a “dimeric” structure analogous tothat formed in a two-chain Fab species. In a further embodiment, thesingle chain humanized antibody fragment is a species comprising a6G4.2.5HV11A variant joined to a 6G4.2.5LV11X variant, 6G4.2.5LV11N35X₃₅variant, 6G4.2.5LV11N35A variant, or 6G4.2.5LV11 by a linker that is tooshort to permit intramolecular pairing of complementary domains, i.e. asingle chain polypeptide monomer that forms a diabody upon dimerizationwith another monomer.

In still another embodiment, the single chain humanized antibodyfragment comprises any pair of light and heavy chains listed in Table 1above joined by means of a flexible peptide linker sequence, wherein theheavy chain and light chain domains can associate in a “dimeric”structure analogous to that formed in a two-chain Fab species. In anadditional embodiment, the single chain humanized antibody fragmentcomprises any pair of light and heavy chains listed in Tables 1–2 abovejoined by a linker that is too short to permit intramolecular pairing ofcomplementary domains, i.e. a single chain polypeptide monomer thatforms a diabody upon dimerization with another monomer.

In yet another embodiment, the invention provides a humanized antibodyfragment comprising a plurality of polypeptide chains, wherein onepolypeptide chain comprises a 6G4.2.5HV11A variant and a secondpolypeptide chain comprises a 6G4.2.5LV11X variant, 6G4.2.5LV11N35X₃₅variant, 6G4.2.5LV11N35A variant, or 6G4.2.5LV11, and the twopolypeptide chains are covalently linked by one or more interchaindisulfide bonds. In a preferred embodiment, the foregoing two-chainantibody fragment is selected from the group consisting of Fab, Fab′,Fab′-SH, and F(ab′)₂.

In an additional embodiment, the invention provides a two-chainhumanized antibody fragment comprising any pair of heavy and lightchains listed in Tables 1–2 above, wherein each chain is contained on aseparate molecule. In another embodiment, the two-chain antibodyfragment comprising any pair of heavy and light chains listed in Tables1–2 above is selected from the group consisting of Fab, Fab′, Fab′-SH,and F(ab′)₂. In a preferred embodiment, the two-chain humanized antibodyfragment is a F(ab′)₂ comprising any pair of heavy and light chainslisted in Tables 1–2 above. In another preferred embodiment, thetwo-chain humanized antibody fragment is a F(ab′)₂ wherein onepolypeptide chain comprises a 6G4.2.51V11A variant and the secondpolypeptide chain comprises the 6G4.2.5LV11N35A.

The invention also provides a humanized antibody or antibody fragmentcomprising a heavy chain containing a 6G4.2.5HV11A variant andoptionally further comprising a light chain containing a 6G4.2.5LV11Xvariant, 6G4.2.5LV11N35X₃₅ variant, 6G4.2.5LV11N35A, or 6G4.2.5HV11,wherein the heavy chain, and optionally the light chain, is (are) fusedto an additional moiety, such as additional immunoglobulin constantdomain sequence. Constant domain sequence can be added to the heavychain and/or light chain sequence(s) to form species with full orpartial length heavy and/or light chain(s). It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species. Preferably,the constant domain sequence is human in origin. Suitable human constantdomain sequences can be obtained from Kabat et al. (supra).

In a preferred embodiment, the humanized antibody or antibody fragmentcomprises a 6G4.2.5HV11A variant in a heavy chain that is fused to orcontains a leucine zipper sequence. The leucine zipper can increase theaffinity or production efficiency of the antibody or antibody fragmentof interest. Suitable leucine zipper sequences include the jun and fosleucine zippers taught by Kostelney et al., J. Immunol., 148: 1547–1553(1992) and the GCN4 leucine zipper described in the Examples below.

C. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forIL-8, the other one is for any other antigen. For example, bispecificantibodies specifically binding a IL-8 and neurotrophic factor, or twodifferent types of IL-8 polypeptides are within the scope of the presentinvention.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published 13 May 1993, and inTraunecker et al., EMBO J. 10:3655 (1991).

According to a different and more preferred approach, antibody-variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant-domain sequences.The fusion preferably is with an immunoglobulin heavy-chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light-chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the maximum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the production of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. This asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. For further details of generating bispecificantibodies, see, for example, Suresh et al., Methods in Enzymology121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217–225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547–1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444–6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

4. Production of Humanized Anti-IL-8 6G4.2.5 Monoclonal Antibody,Antibody Fragments, and Variants

The antibodies and antibody fragments of the invention can be producedusing any convenient antibody manufacturing process known in the art.Typically, the antibody or antibody fragment is made using recombinantexpression systems. A multiple polypeptide chain antibody or antibodyfragment species can be made in a single host cell expression systemwherein the host cell produces each chain of the antibody or antibodyfragment and assembles the polypeptide chains into a multimericstructure to form the antibody or antibody fragment in vivo, followed byrecovery of the antibody or antibody fragment from the host cell. Forexample, suitable recombinant expression systems for the production ofcomplete antibody or antibody fragment are described in Lucas et al.,Nucleic Acids Res., 24: 1774–1779 (1996). Alternatively, the separatepolypeptide chains of the desired antibody or antibody fragment can bemade in separate expression host cells, separately recovered from therespective host cells, and then mixed in vitro under conditionspermitting the formation of the multi-subunit antibody or antibodyfragment of interest. For example, U.S. Pat. No. 4,816,567 to Cabilly etal. and Carter et al., Bio/Technology, 10: 163–167 (1992) providemethods for recombinant production of antibody heavy and light chains inseparate expression hosts followed by assembly of antibody from separateheavy and light chains in vitro.

The following discussion of recombinant expression methods appliesequally to the production of single chain antibody polypeptide speciesand multi-subunit antibody and antibody fragment species. Allrecombinant procedures for the production of antibody or antibodyfragment provided below shall be understood to describe: (1) manufactureof single chain antibody species as the desired end-product; (2)manufacture of multi-subunit antibody or antibody fragment species byproduction of all subunits in a single host cell, subunit assembly inthe host cell, optionally followed by host cell secretion of themulti-subunit end-product into the culture medium, and recovery of themulti-subunit end-product from the host cell and/or culture medium; and(3) manufacture of multi-subunit antibody or antibody fragment byproduction of subunits in separate host cells (optionally followed byhost cell secretion of subunits into the culture medium), recovery ofsubunits from the respective host cells and/or culture media, followedby in vitro subunit assembly to form the multi-subunit end-product. Inthe case of a multi-subunit antibody or antibody fragment produced in asingle host cell, it will be appreciated that production of the varioussubunits can be effected by expression of multiple polypeptide-encodingnucleic acid sequences carried on a single vector or by expression ofpolypeptide-encoding nucleic acid sequences carried on multiple vectorscontained in the host cell.

A. Construction of DNA Encoding Humanized 6G4.2.5 Monoclonal AntibodiesAntibody Fragments, and Variants

Following the selection of the humanized antibody or antibody fragmentof the invention according to the methods described above, thepractitioner can use the genetic code to design DNAs encoding thedesired antibody or antibody fragment. In one embodiment, codonspreferred by the expression host cell are used in the design of a DNAencoding the antibody or antibody fragment of interest. DNA encoding thedesired antibody or antibody fragment can be prepared by a variety ofmethods known in the art. These methods include, but are not limited to,chemical synthesis by any of the methods described in Engels et al.,Agnew. Chem. Int. Ed. Engl., 28: 716–734 (1989), the entire disclosureof which is incorporated herein by reference, such as the triester,phosphite, phosphoramidite and H-phosphonate methods.

A variation on the above procedures contemplates the use of genefusions, wherein the gene(s) encoding the antibody or antibody fragmentis associated, in the vector, with a gene encoding another protein or afragment of another protein. This results in the antibody or antibodyfragment being produced by the host cell as a fusion with anotherprotein. The “other” protein is often a protein or peptide which can besecreted by the cell, making it possible to isolate and purify thedesired protein from the culture medium and eliminating the necessity ofdestroying the host cells which arises when the desired protein remainsinside the cell. Alternatively, the fusion protein can be expressedintracellularly. It is advantageous to use fusion proteins that arehighly expressed.

The use of gene fusions, though not essential, can facilitate theexpression of heterologous proteins in E. coli as well as the subsequentpurification of those gene products (Harris, T. J. R. in GeneticEngineering, Williamson, R., Ed., Academic, London, Vol. 4, p.127(1983); Uhlen, M. & Moks, T., Methods Enzymol. 185:129–143 (1990)).Protein A fusions are often used because the binding of protein A, ormore specifically the Z domain of protein A, to IgG provides an“affinity handle” for the purification of the fused protein (Nilsson. B.& Abrahmsen, L. Methods Enzymol. 185:144–161 (1990)). It has also beenshown that many heterologous proteins are degraded when expresseddirectly in E. coli, but are stable when expressed as fusion proteins(Marston, F. A. O., Biochem J. 240: 1 (1986)).

Fusion proteins can be cleaved using chemicals, such as cyanogenbromide, which cleaves at a methionine, or hydroxylamine, which cleavesbetween an Asn and Gly. Using standard recombinant DNA methodology, thenucleotide base pairs encoding these amino acids may be inserted justprior to the 5′ end of the antibody or antibody fragment gene(s).

Alternatively, one can employ proteolytic cleavage of fusion proteins,which has been recently reviewed (Carter, P. (1990) in ProteinPurification: From Molecular Mechanisms to Large-Scale Processes,Ladisch, M. R., Willson, R. C., Painton, C. C., and Builder, S. E.,eds., American Chemical Society Symposium Series No. 427, Ch 13,181–193).

Proteases such Factor Xa, thrombin, subtilisin and mutants thereof, havebeen successfully used to cleave fusion proteins. Typically, a peptidelinker that is amenable to cleavage by the protease used is insertedbetween the “other” protein (e.g., the Z domain of protein A) and theprotein of interest, such as humanized anti-IL-8 antibody or antibodyfragment. Using recombinant DNA methodology, the nucleotide base pairsencoding the linker are inserted between the genes or gene fragmentscoding for the other proteins. Proteolytic cleavage of the partiallypurified fusion protein containing the correct linker can then becarried out on either the native fusion protein, or the reduced ordenatured fusion protein.

Various techniques are also available which may now be employed toproduce variant humanized antibodies or antibody fragments, whichencodes for additions, deletions, or changes in amino acid sequence ofthe resultant protein(s) relative to the parent humanized antibody orantibody fragment.

By way of illustration, with expression vectors encoding humanizedantibody or antibody fragment in hand, site specific mutagenesis (Kunkelet al., Methods Enzymol. 204:125–139 (1991); Carter, P., et al., Nucl.Acids. Res. 13:4331 (1986); Zoller, M. J. et al., Nucl. Acids Res.10:6487 (1982)), cassette mutagenesis (Wells, J. A., et al., Gene 34:315(1985)), restriction selection mutagenesis (Wells, J. A., et al.,Philos. Trans, R. Soc. London SerA 317, 415 (1986)) or other knowntechniques may be performed on the antibody or antibody fragment DNA.The variant DNA can then be used in place of the parent DNA by insertioninto the aforementioned expression vectors. Growth of host bacteriacontaining the expression vectors with the mutant DNA allows theproduction of variant humanized antibodies or antibody fragments, whichcan be isolated as described herein.

B. Insertion of DNA into a Cloning Vehicle

The DNA encoding the antibody or antibody fragment is inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. Many vectors are available, and selection of the appropriatevector will depend on (1) whether it is to be used for DNA amplificationor for DNA expression, (2) the size of the DNA to be inserted into thevector, and (3) the host cell to be transformed with the vector. Eachvector contains various components depending on its function(amplification of DNA or expression of DNA) and the host cell for whichit is compatible. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(i) Signal Sequence Component

In general, a signal sequence may be a component of the vector, or itmay be a part of the antibody or antibody fragment DNA that is insertedinto the vector. Preferably, a heterologous signal sequence selected andfused to the antibody or antibody fragment DNA such that the signalsequence in the corresponding fusion protein is recognized, transportedand processed (i.e., cleaved by a signal peptidase) in the host cell'sprotein secretion system. In the case of prokaryotic host cells, thesignal sequence is selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.In a preferred embodiment, the STII signal sequence is used as describedin the Examples below. For yeast secretion the native signal sequencemay be substituted by, e.g., the yeast invertase leader, α factor leader(including, Saccharomyces and Kluyveromyces α-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is homologous to a sequencefound in Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theantibody or antibody fragment DNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, ortetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g. the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327(1982)), mycophenolic acid (Mulligan et al., Science, 209: 1422 (1980))or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410–413 (1985)). Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug (G418 or neomycin(geneticin), xgpt (mycophenolic acid), and hygromycin, respectively.)

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody or antibody fragment nucleic acid, such as dihydrofolatereductase (DHFR) or thymidine kinase. The mammalian cell transformantsare placed under selection pressure which only the transformants areuniquely adapted to survive by virtue of having taken up the marker.Selection pressure is imposed by culturing the transformants underconditions in which the concentration of selection agent in the mediumis successively changed, thereby leading to amplification of both theselection gene and the DNA that encodes the antibody or antibodyfragment. Amplification is the process by which genes in greater demandfor the production of a protein critical for growth are reiterated intandem within the chromosomes of successive generations of recombinantcells. Increased quantities of the antibody or antibody fragment aresynthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216 (1980). The transformed cells are then exposed toincreased levels of methotrexate. This leads to the synthesis ofmultiple copies of the DHFR gene, and, concomitantly, multiple copies ofother DNA comprising the expression vectors, such as the DNA encodingthe antibody or antibody fragment. This amplification technique can beused with any otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1,notwithstanding the presence of endogenous DHFR if, for example, amutant DHFR gene that is highly resistant to Mtx is employed (EP117,060). Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding the antibody or antibody fragment, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3′phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7. Stinchcomb et al., Nature, 282: 39 (1979);Kingsman et al., Gene, 7: 141 (1979); or Tschemper et al., Gene, 10: 157(1980). The trp1 gene provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44076 or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors usually contain a promoter that is recognized by thehost organism and is operably linked to the antibody or antibodyfragment nucleic acid. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofa particular nucleic acid sequence, such as the antibody or antibodyfragment encoding sequence, to which they are operably linked. Suchpromoters typically fall into two classes, inducible and constitutive.Inducible promoters are promoters that initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g. the presence or absence of a nutrient or achange in temperature. At this time a large number of promotersrecognized by a variety of potential host cells are well known.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615(1978); and Goeddel et al., Nature, 281: 544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 (1980) and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21–25 (1983)).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled worker tooperably ligate them to DNA encoding the antibody or antibody fragment(Siebenlist et al., Cell, 20: 269 (1980)) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also generally will contain a Shine-Dalgamo (S.D.) sequenceoperably linked to the DNA encoding the antibody or antibody fragment.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg., 7: 149 (1968); and Holland, Biochemistry, 17: 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

Vector driven transcription of antibody or antibody fragment encodingDNA in mammalian host cells can be controlled by promoters obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and most preferably Simian Virus 40(SV40), from heterologous mammalian promoters, e.g. the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273: 113 (1978); Mulligan andBerg, Science, 209: 1422–1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA, 78: 7398–7402 (1981). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway et al., Gene, 18: 355–360 (1982). Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso Gray et al., Nature, 295: 503–508 (1982) on expressing cDNAencoding immune interferon in monkey cells, Reyes et al., Nature, 297:598–601 (1982) on expression of human-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus, Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79: 5166–5170(1982) on expression of the human interferon 1 gene in cultured mouseand rabbit cells, and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777–6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding antibody or antibody fragment by highereukaryotic host cells is often increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10–300 bp, that act on a promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent having been found 5′ (Laimins et al., Proc. Natl. Acad. Sci.USA, 78: 993 (1981)) and 3′ (Lusky et al., Mol. Cell Bio., 3: 1108(1983)) to the transcription unit, within an intron (Banerji et al.,Cell, 33: 729 (1983)) as well as within the coding sequence itself(Osborne et al., Mol. Cell Bio., 4: 1293 (1984)). Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,-fetoprotein and insulin). Typically, however, one will use an enhancerfrom a eukaryotic cell virus. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100–270), the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. See also Yaniv, Nature,297: 17–18 (1982) on enhancing elements for activation of eukaryoticpromoters. The enhancer may be spliced into the vector at a position 5′or 3′ to the antibody or antibody fragment DNA but is preferably locatedat a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) can also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody or antibody fragment. The 3′untranslated regions also include transcription termination sites.

Suitable vectors containing one or more of the above listed componentsand the desired coding and control sequences are constructed by standardligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the antibody or antibody fragment. In general, transientexpression involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the antibody or antibody fragment in recombinant vertebratecell culture are described in Gething et al., Nature, 293: 620–625(1981); Mantei et al., Nature, 281: 40–46 (1979); Levillson et al., EP17,060; and EP 117,058. A particularly useful plasmid for mammalian cellculture expression of the IgE peptide antagonist is pRK5 (EP pub. no.307,247) or pSVI6B (PCT pub. no. WO 91/08291 published 13 Jun. 1991).

C. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryote cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescens. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coli 1776(ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes. In a preferredembodiment, the E. coli strain 49D6 is used as the expression host asdescribed in the Examples below. Review articles describing therecombinant production of antibodies in bacterial host cells includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs., 130: 151 (1992).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors containing antibody orantibody fragment DNA. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as S. pombe (Beach andNurse, Nature, 290: 140 (1981)), Kluyveromyces lactis (Louvencourt etal., J. Bacteriol., 737 (1983)), yarrowia (EP 402,226), Pichia pastoris(EP 183,070), Trichoderma reesia (EP 244,234), Neurospora crassa (Caseet al., Proc. Natl. Acad. Sci. USA, 76: 5259–5263 (1979)), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112: 284–289 (1983); Tilburn et al., Gene, 26:205–221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470–1474(1984)) and A. niger (Kelly and Hynes, EMBO J., 4: 475–479 (1985)).

Host cells derived from multicellular organisms can also be used in therecombinant production of antibody or antibody fragment. Such host cellsare capable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori host cells have been identified. See, e.g., Luckow et al.,Bio/Technology, 6: 47–55 (1988); Miller et al., in Genetic Engineering,Setlow, J. K. et al., 8: 277–279 (Plenum Publishing, 1986), and Maeda etal., Nature, 315: 592–594 (1985). A variety of such viral strains arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the antibody or antibody fragment DNA. During incubation of theplant cell culture with A. tumefaciens, the DNA encoding antibody orantibody fragment is transferred to the plant cell host such that it istransfected, and will, under appropriate conditions, express theantibody or antibody fragment DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1: 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published 21 Jun. 1989.

Vertebrate cell culture is preferred for the recombinant production offull length antibodies. The propagation of vertebrate cells in culture(tissue culture) has become a routine procedure in recent years (TissueCulture, Academic Press, Kruse and Patterson, editors (1973)). Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCCCCL10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23: 243–251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (WI 38, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y. Acad. Sci., 383: 44–68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma cell line (Hep G2). Preferred host cellsare human embryonic kidney 293 and Chinese hamster ovary cells. Myelomacells that do not otherwise produce immunoglobulin protein are alsouseful host cells for the recombinant production of full lengthantibodies.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 published29 Jun. 1989. For mammalian cells without such cell walls, the calciumphosphate precipitation method described in sections 16.30–16.37 ofSambrook et al., supra, is preferred. General aspects of mammalian cellhost system transformations have been described by Axel in U.S. Pat. No.4,399,216 issued 16 Aug. 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen et al., J. Bact.,130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829(1979). However, other methods for introducing DNA into cells such as bynuclear injection, electroporation, or by protoplast fusion may also beused.

D. Culturing the Host Cells

Prokaryotic cells used to produce the antibody or antibody fragment arecultured in suitable media as described generally in Sambrook et al.,supra.

The mammalian host cells used to produce the antibody or antibodyfragment can be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz., 58: 44(1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of allof which are incorporated herein by reference, may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

E. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77: 5201–5205 (1980)), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³²P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product, where the labels are usually visually detectable, such asenzymatic labels, fluorescent labels, luminescent labels, and the like.A particularly sensitive staining technique suitable for use in thepresent invention is described by Hsu et al., Am. J. Clin. Path., 75:734–738 (1980).

F. Purification of the Antibody or Antibody Fragment

In the case of a host cell secretion system, the antibody or antibodyfragment is recovered from the culture medium. Alternatively, theantibody can be produced intracellularly, or produced in the periplasmicspace of a bacterial host cell. If the antibody is producedintracellularly, as a first step, the host cells are lysed, and theresulting particulate debris is removed, for example, by centrifugationor ultrafiltration. Carter et al., Bio/Technology 10:163–167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the antibody is secreted into themedium, supernatants from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1–13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin Sepharose™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5–4.5, preferably performed at low salt concentrations(e.g. from about 0–0.25M salt).

G. Production of Antibody Fragments

Various techniques have been developed for the production of thehumanized antibody fragments of the invention, including Fab, Fab′,Fab′-SH, or F(ab′)₂ fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., Journal of Biochemical and Biophysical Methods24:107–117 (1992) and Brennan et al., Science, 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.For example, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology, 10:163–167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner.

5. Uses of Anti-IL-8 Antibodies

A. Diagnostic Uses

For diagnostic applications requiring the detection or quantitation ofIL-8, the antibodies or antibody fragments of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g.,¹²⁵I, ³²P, ¹⁴C, or ³H; or an enzyme, such as alkaline phosphatase,beta-galactosidase, or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody orantibody fragment to the detectable moiety can be employed, includingthose methods described by Hunter et al., Nature 144:945 (1962); Davidet al., Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth.40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies and antibody fragments of the present invention can beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays. Forexample, see Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147–158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which can be a IL-8 or an immunologically reactive portion thereof) tocompete with the test sample analyte (IL-8) for binding with a limitedamount of antibody or antibody fragment. The amount of IL-8 in the testsample is inversely proportional to the amount of standard that becomesbound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies or antibody fragmentsgenerally are insolubilized before or after the competition, so that thestandard and analyte that are bound to the antibodies can convenientlybe separated from the standard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different antigenic portion, or epitope, of the protein(IL-8) to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex (U.S. Pat. No. 4,376,110). The secondantibody can itself be labeled with a detectable moiety (direct sandwichassays) or can be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme (e.g., horseradish peroxidase).

IL-8 antibodies and antibody fragments also are useful for the affinitypurification of IL-8 from recombinant cell culture or natural sources.For example, these antibodies can be fixed to a solid support bytechniques well known in the art so as to purify IL-8 from a source suchas culture supernatant or tissue.

B. Therapeutic Compositions and Administration of Anti-IL-8 Antibody

The humanized anti-IL-8 antibodies and antibody fragments of theinvention are useful in the treatment of inflammatory disorders,including inflammations of the lung, such as adult respiratory distresssyndrome (ARDS) and any stage of acute lung injury in the pathogenesisof ARDS described in Bernard et al., Am. J. Respir. Crit. Care Med.,149: 818–824 (1994), bacterial pneumonia, hypovolemic shock, ischemicreperfusion disorders such as surgical tissue reperfusion injury,myocardial ischemic conditions such as myocardial infarction,reperfusion after cardiac surgery, cardiac arrest, and constrictionafter percutaneous transluminal coronary angioplasty, inflammatory boweldisorders such is ulcerative colitis, and autoimmune diseases such asrheumatoid arthritis. In addition, the humanized anti-IL-8 antibodiesand antibody fragments of the invention are useful in the treatment ofasthmatic diseases, such as allergic asthma.

Therapeutic formulations of the humanized anti-IL-8 antibodies andantibody fragments are prepared for storage by mixing the antibody orantibody fragment having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, supra), in the form of lyophilizedcake or aqueous solutions. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

The humanized anti-IL-8 mAb or antibody fragment to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The humanized anti-IL-8 mAb orantibody fragment ordinarily will be stored in lyophilized form or insolution.

Therapeutic humanized anti-IL-8 mAb or antibody fragment compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The route of humanized anti-IL-8 mAb or antibody fragment administrationis in accord with known methods, e.g., inhalation, injection or infusionby intravenous, intraperitoneal, intracerebral, intramuscular,intraocular, intraarterial, or intralesional routes, by enema orsuppository, or by sustained release systems as noted below. Preferablythe antibody is given systemically or at a site of inflammation.

In one embodiment, the invention provides for the treatment of asthmaticdiseases by administration of humanized anti-IL-8 mAb or antibodyfragment to the respiratory tract. The invention contemplatesformulations comprising humanized anti-IL-8 mAb or antibody fragment foruse in a wide variety of devices that are designed for the delivery ofpharmaceutical compositions and therapeutic formulations to therespiratory tract. In one aspect, humanized anti-IL-8 mAb or antibodyfragment is administered in aerosolized or inhaled form. The humanizedanti-IL-8 mAb or antibody fragment, combined with a dispersing agent, ordispersant, can be administered in an aerosol formulation as a drypowder or in a solution or suspension with a diluent.

Suitable dispersing agents are well known in the art, and include butare not limited to surfactants and the like. Surfactants are generallyused in the art to reduce surface induced aggregation of protein causedby atomization of the solution forming the liquid aerosol. Examples ofsuch surfactants include polyoxyethylene fatty acid esters and alcohols,and polyexyethylene sorbitan fatty acid esters. Amounts of surfactantsused will vary, being generally within the range of about 0.001 to 4% byweight of the formulation. In a specific aspect, the surfactant ispolyoxyethylene sorbitan monooleate or sorbitan trioleate.

Liquid aerosol formulations contain the humanized anti-IL-8 mAb orantibody fragment and a dispersing agent in a physiologically acceptablediluent. The dry powder formulations of the invention consist of afinely divided solid form of the humanized anti-IL-8 mAb or antibodyfragment and a dispersing agent, and optionally a bulking agent, such aslactose, sorbitol, sucrose, or mannotil, and the like, to facilitatedispersal of the powder. With either the liquid or dry powder aerosolformulation, the formulations must be aerosolized. It must be brokendown into liquid or solid particles in order to ensure that theaerosolized dose actually reaches the bronchii and/or alveoli, asdesired. For example, in the methods for treatment of asthma providedherein, it is preferable to deliver aerosolized humanized anti-IL-8 mAbor antibody fragment to the bronchii. In other embodiments, such as thepresent methods for treating ARDS and any stage of acute lung injury inthe pathogenesis of ARDS, it is preferable to deliver aerosolizedhumanized anti-IL-8 mAb or antibody fragment to the alveoli. In general,the mass median dynamic diameter will be 5 micrometers (μm) or less toensure that the drug particles reach the lung bronchii or alveoli(Wearly, L. L., 1991, Crit. Rev. in Ther. Drug Carrier Systems, 8:333).

With regard to construction of the delivery device, any form ofaerosolization known in the art, including but not limited tonebulization, atomization or pump aerosolization of a liquidformulation, and aerosolization of a dry powder formulation, can be usedin the practice of the invention. A delivery device that is uniquelydesigned for administration of solid formulations is envisioned. Often,the aerosolization of a liquid or a dry powder formulation will requirea propellent. The propellent can be any propellent generally used in theart. Examples of useful propellants include cholorofluorocarbons,hydrofluorocarbons, hydrochlorofluorocarbons, and hydrocarbons,including trifluoromethane, dichlorofluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, andcombinations thereof.

In a preferred aspect of the invention, the device for aerosolization isa metered dose inhaler. A metered dose inhaler provides a specificdosage when administered, rather than a variable dose depending onadministration. Such a metered dose inhaler can be used with either aliquid or a dry powder aerosol formulation.

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197–22and can be used in connection with the present invention.

Sustained release systems can be used in the practice of the methods ofthe invention. Suitable examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers 22:547 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.15:167 (1981) and Langer, Chem. Tech. 12:98 (1982)), ethylene vinylacetate (Langer et al., supra) or poly-D-(−)-3-hydroxybutyric acid (EP133,988). Sustained-release humanized anti-IL-8 antibody or antibodyfragment compositions also include liposomally entrapped antibody orantibody fragment. Liposomes containing an antibody or antibody fragmentare prepared by methods known per se: DE 3,218,121; Epstein et al.,Proc. Natl. Acad. Sci. U.S.A. 82:3688 (1985); Hwang et al., Proc. Natl.Acad. Sci. U.S.A. 77:4030 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83–118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200–800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mole percent cholesterol, theselected proportion being adjusted for the most efficacious antibody orantibody fragment therapy.

An “effective amount” of the humanized anti-IL-8 antibody or antibodyfragment to be employed therapeutically will depend, for example, uponthe therapeutic objectives, the route of administration, and thecondition of the patient. Accordingly, it will be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. Typically, theclinician will administer the humanized anti-IL-8 antibody or antibodyfragment until a dosage is reached that achieves the desired effect. Theprogress of this therapy is easily monitored by conventional assays.

In the treatment and prevention of an inflammatory disorder or asthmaticdisorder with a humanized anti-IL-8 antibody or antibody fragment of theinvention, the antibody composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the antibody, the particular type of antibody, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of antibody to be administered will be governed by such considerations,and is the minimum amount necessary to prevent, ameliorate, or treat theinflammatory disorder, including treating acute or chronic respiratorydiseases and reducing inflammatory responses. Such amount is preferablybelow the amount that is toxic to the host or renders the hostsignificantly more susceptible to infections.

As a general proposition, the initial pharmaceutically effective amountof the antibody or antibody fragment administered parenterally per dosewill be in the range of about 0.1 to 50 mg/kg of patient body weight perday, with the typical initial range of antibody used being 0.3 to 20mg/kg/day, more preferably 0.3 to 15 mg/kg/day.

In one embodiment, using systemic administration, the initialpharmaceutically effective amount will be in the range of about 2 to 5mg/kg/day.

For methods of the invention using administration by inhalation, theinitial pharmaceutically effective amount will be in the range of about1 microgram (μg)/kg/day to 100 mg/kg/day.

The invention provides for both prophylactic and therapeutic treatmentof inflammatory disorders. Without intending to limit the methods of theinvention to a particular mechanism of action or a particular diseaseintervention strategy, it is noted that in some indications it isbeneficial to treat the disease in question prior to or early on in thestage of the underlying disease that involves neutrophil activation,recruitment and infiltration at sites of inflammation. Accordingly, itmay be advantageous to utilize the humanized anti-IL-8 mAb or antibodyfragment in a prophylactic treatment regimen for an inflammatory diseaseindication in order to attenuate or eliminate a pathogenic neutrophilresponse that may or will arise during the course of the disease.

In patients at risk of developing acute lung injury with possible orlikely progression to ARDS, it is desirable to employ a prophylacticcourse of treatment in order to ameliorate or prevent the deteriorationof lung function and the pathogenesis of associated disease sequelae(which may greatly increase patient morbidity and mortality) prior tothe onset of such conditions. Certain biological parameters, such asIL-8 levels in bronchial alveolar lavage (BAL) fluid and ferritin levelsin serum, can be used for prognosis of acute lung injury and ARDS inpatients who are predisposed to such disease progression, i.e. patientssuffering from diseases or other insults that commonly precipitate acutelung injury and ARDS, such as aspiration, diffuse pulmonary infection,near-drowning, toxic inhalation, lung contusion, multiple trauma,pancreatitis, perforated bowel, sepsis, and the like. In one embodiment,acute lung injury and ARDS at-risk patients presenting BAL fluid IL-8concentrations of at or above 0.2 ng/ml are selected for prophylactictreatment according to the methods of the invention. Any suitable methodfor assay of IL-8 in patient BAL fluid may be employed, such as themethod described in Donnelly et al., Lancet, 341: 643–647 (1993).

In another embodiment, acute lung injury/ARDS at-risk female and malepatients presenting ferritin serum concentrations of at or above 270ng/ml and 680 ng/ml, respectively, are selected for prophylactictreatment according to the methods of the invention. Any suitable methodfor assay of ferritin in patient serum may be employed, such as themethod described in U.S. Pat. No. 5,679,532 for “Serum Ferritin as aPredictor of the Acute Respiratory Distress Syndrome” to Repine issuedon Oct. 21, 1997.

In patients presenting ischemic conditions or undergoing surgicalprocedures that generate ischemic conditions in tissue and concomitantrisk of tissue injury upon reperfusion, it is desirable to employ acourse of treatment wherein the humanized anti-IL-8 mAb or antibodyfragment is administered to the patient prior to the reperfusion ofischemic tissue, or prior to or as soon as possible after the onset ofan inflammatory response following reperfusion of ischemic tissue. Inthe patients presenting acute myocardial infarction, for example, it isadvantageous to employ a course of treatment wherein the humanizedanti-IL-8 mAb or antibody fragment is administered to the patient priorto or concomitant with recanalization therapy, including pharmaceuticalrecanalization therapies such as the administration of tissueplasminogen activators, streptokinase, or other thrombolytic drugs withor without anti-clotting agents such as platelet-fibrin bindingantagonists (e.g. anti-IIbIIIa integrin antibody), blood thinning agentssuch as heparin, or other anti-reocclusion agents such as aspirin, andthe like, and including mechanical recanalization therapies such aspercutaneous transluminal coronary angioplasty, or wherein the humanizedanti-IL-8 mAb or antibody fragment is administered to the patient priorto or as soon as possible after the onset of an inflammatory responsefollowing reperfusion of ischemic myocardium. In yet another embodiment,the humanized anti-IL-8 mAb or antibody fragment of the invention can beemployed in the methods of treating acute myocardial infarction withanti-IL-8 antibody described in WO 97/40215 published Oct. 30, 1997.

The invention provides for both prophylactic and therapeutic treatmentof asthma with humanized anti-IL-8 mAb and antibody fragment. In thecase of prophylactic treatment for allergic asthma with the antibodiesor antibody fragments of the invention, it is desirable to administerabout 0.1 to 10 mg/kg of the antibody agent to the patient up to about24 hours prior to anticipated exposure to allergen or prior to onset ofallergic asthma. In the case of therapeutic treatment for acute asthma,including allergic asthma, it is desirable to treat the asthmaticpatient as early as possible following onset of an asthma attack. In oneembodiment, an episode of acute asthma is treated within 24 hours of theonset of symptoms by administration of about 0.1 to 10 mg/kg of ananti-IL-8 antibody agent. However, it will be appreciated that themethods of the invention can be used to ameliorate symptoms at any pointin the pathogenesis of asthmatic disease. Additionally, the methods ofthe invention can be used to alleviate symptoms of chronic asthmaticconditions.

The antibody or antibody fragment need not be, but is optionallyformulated with one or more agents currently used to prevent or treatthe inflammatory disorder or asthmatic disease in question. For example,in rheumatoid arthritis, the antibody can be given in conjunction with aglucocorticosteroid. In the case of treating asthmatic diseases withanti-IL-8 antibody or antibody fragment, the invention contemplates thecoadministration of antibody or antibody fragment and one or moreadditional agents useful in treating asthma, such as bronchodilators,antihistamines, epinephrine, and the like. The effective amount of suchother agents depends on the amount of antibody or antibody fragmentpresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all references cited in thespecification, and the disclosures of all citations in such references,are expressly incorporated herein by reference.

EXAMPLES

A. Generation and Characterization of Monoclonal Antibodies AgainstHuman IL-8

Balb/c mice were immunized in each hind footpad or intraperitoneallywith 10 μg of recombinant human IL-8 (produced as a fusion of(ser-IL-8)₇₂ with ubiquitin (Hebert et al. J. Immunology 145:3033–3040(1990)); IL-8 is available commercially from PeproTech, Inc., RockyHill, N.J.) resuspended in MPL/TDM (Ribi Immunochem. Research Inc.,Hamilton, Mont.) and boosted twice with the same amount of IL-8. Inthese experiments, “IL-8” is intended to mean (ser-IL-8)₇₂ unlessotherwise specified. A final boost of 10 μg of IL-8 was given 3 daysbefore the fusion. Spleen cells or popliteal lymph node cells were fusedwith mouse myeloma P3X63Ag8U.1 (ATCC CRL1597), a non-secreting clone ofthe myeloma P3X63Ag8, using 35% polyethylene glycol as described before.Ten days after the fusion, culture supernatant was screened for thepresence of monoclonal antibodies to IL-8 by ELISA.

The ELISA was performed as follows. Nunc 96-well immunoplates (Flow Lab,McLean, Va.) were coated with 50 μl/well of 2 μg/ml IL-8 inphosphate-buffered saline (PBS) overnight at 4° C. The remaining stepswere carried out at room temperature. Nonspecific binding sites wereblocked with 0.5% bovine serum albumin (BSA) for 1 hour (hr). Plateswere then incubated with 50 μl/well of hybridoma culture supernatantsfrom 672 growing parental fusion wells for 1 hr, followed by theincubation with 50 μl/well of 1:1000 dilution of a 1 mg/ml stocksolution of alkaline phosphatase-conjugated goat anti-mouse Ig (TagoCo., Foster City, Calif.) for 1 hr. The level of enzyme-linked antibodybound to the plate was determined by the addition of 100 μl/well of 0.5mg/ml of r-nitrophenyl phosphate in sodium bicarbonate buffer, pH 9.6.The color reaction was measured at 405 nm with an ELISA plate reader(Titertrek Multiscan, Flow Lab, McLean, Va.). Between each step, plateswere washed three times in PBS containing 0.05% Tween 20.

Culture supernatants which promoted 4-fold more binding of IL-8 than didcontrol medium were selected as positives. According to this criterion,16 of 672 growing parental fusion wells (2%) were positive. Thesepositive hybridoma cell lines were cloned at least twice by using thelimiting dilution technique.

Seven of the positive hybridomas were further characterized as follows.The isotypes of the monoclonal antibodies were determined by coatingNunc 96-well immunoplates (Flow Lab, McLean, Va.) with IL-8 overnight,blocking with BSA, incubating with culture supernatants followed by theaddition of predetermined amount of isotype-specific alkalinephosphatase-conjugated goat anti-mouse Ig (Fisher Biotech, Pittsburgh,Pa.). The level of conjugated antibodies bound to the plate wasdetermined by the addition of r-nitrophenyl phosphate as describedabove.

All the monoclonal antibodies tested belonged to either IgG, or IgG₂immunoglobulin isotype. Ascites fluid containing these monoclonalantibodies had antibody titers in the range of 10,000 to 100,000 asdetermined by the reciprocal of the dilution factor which gave 50% ofthe maximum binding in the ELISA.

To assess whether these monoclonal antibodies bound to the sameepitopes, a competitive binding ELISA was performed. At a ratio ofbiotinylated mAb to unlabeled mAb of 1:100, the binding of biotinylatedmAb 5.12.14 was significantly inhibited by its homologous mAb but not bymAb 4.1.3, while the binding of biotinylated mAb 4.1.3 was inhibited bymAb 4.1.3 but not by mAb 5.12.14. Monoclonal antibody 5.2.3 behavedsimilarly to mAb 4.1.3, while monoclonal antibodies 4.8 and 12.3.9 weresimilar to mAb 5.12.14. Thus, mAb 4.1.3 and mAb 5.2.3 bind to adifferent epitope(s) than the epitope recognized by monoclonalantibodies 12.3.9, 4.8 and 5.12.14.

Immunodot blot analysis was performed to assess antibody reactivity toIL-8 immobilized on nitrocellulose paper. All seven antibodiesrecognized IL-8 immobilized on paper, whereas a control mouse IgGantibody did not.

The ability of these monoclonal antibodies to capture soluble ¹²⁵I-IL-8was assessed by a radio immune precipitation test (RIP). Briefly, tracer¹²⁵I-IL-8 (4×10⁴ cpm) was incubated with various dilutions of themonoclonal anti-IL-8 antibodies in 0.2 ml of PBS containing 0.5% BSA and0.05% Tween 20 (assay buffer) for 1 hr at room temperature. One hundredmicroliters of a predetermined concentration of goat anti-mouse Igantisera (Pel-Freez, Rogers, Ark.) were added and the mixture wasincubated at room temperature for 1 hr. Immune complexes wereprecipitated by the addition of 0.5 ml of 6% polyethylene glycol (M.W.8000) kept at 4° C. After centrifugation at 2,000×g for 20 min at 4° C.,the supernatant was removed by aspiration and the radioactivityremaining in the pellet was counted in a gamma counter. Percent specificbinding was calculated as (precipitated cpm−background cpm)/(totalcpm−background cpm). Monoclonal antibodies 4.1.3, 5.2.3, 4.8, 5.12.14and 12.3.9 captured ¹²⁵I-IL-8 very efficiently, while antibodies 9.2.4and 8.9.1 were not able to capture soluble ¹²⁵I-IL-8 in the RIP eventhough they could bind to IL-8 coated onto ELISA plates (Table I).

The dissociation constants of these monoclonal antibodies weredetermined using a competitive binding RIP assay. Briefly, competitiveinhibition of the binding each antibody to ¹²⁵I-IL-8 (20,000–40,000 cpmper assay) by various amounts of unlabeled IL-8 was determined by theRIP described above. The dissociation constant (affinity) of each mAbwas determined by using Scatchard plot analysis (Munson, et al., Anal.Biochem. 107:220 (1980)) as provided in the VersaTerm-PRO computerprogram (Synergy Software, Reading, Pa.). The K_(d)'s of thesemonoclonal antibodies (with the exception of 9.2.4. and 8.9.1) were inthe range from 2×10⁻⁸ to 3×10⁻¹⁰ M. Monoclonal antibody 5.12.14 with aK_(d) of 3×10⁻¹⁰ M showed the highest affinity among all the monoclonalantibodies tested (Table 3).

TABLE 3 Characterization of Anti-IL-8 Monoclonal Antibodies % SpecificBinding Antibody to IL-8 K_(d) (M) Isotype pI 4.1.3 58 2 × 10⁻⁹ IgG₁4.3–6.1 5.2.3 34 2 × 10⁻⁸ IgG₁ 5.2–5.6 9.2.4 1 — IgG₁ 7.0–7.5 8.9.1 2 —IgG₁ 6.8–7.6 4.8 62 3 × 10⁻⁸ IgG_(2a) 6.1–7.1 5.12.14 98  3 × 10⁻¹⁰IgG_(2a) 6.2–7.4 12.3.9 86 2 × 10⁻⁹ IgG_(2a) 6.5–7.1

To assess the ability of these monoclonal antibodies to neutralize IL-8activity, the amount of ¹²⁵I-IL-8 bound to human neutrophils in thepresence of various amounts of culture supernatants and purifiedmonoclonal antibodies was measured. Neutrophils were prepared by usingMono-Poly Resolving Medium (M-PRM) (Flow Lab. Inc., McLean. Va.).Briefly fresh, heparinized human blood was loaded onto M-PRM at a ratioof blood to medium, 3.5:3.0, and centrifuged at 300×g for 30 min at roomtemperature. Neutrophils enriched at the middle layer were collected andwashed once in PBS. Such a preparation routinely contained greater than95% neutrophils according to the Wright's Giemsa staining. The receptorbinding assay was done as follows. 50 μl of ¹²⁵I-IL-8 (5 ng/ml) wasincubated with 50 μl of unlabeled IL-8 (100 μg/ml) or monoclonalantibodies in PBS containing 0.1% BSA for 30 min at room temperature.The mixture was then incubated with 100 μl of neutrophils (10′ cells/ml)for 15 min at 37° C. The ¹²⁵I-IL-8 bound was separated from the unboundmaterial by loading mixtures onto 0.4 ml of PBS containing 20% sucroseand 0.1% BSA and by centrifugation at 300×g for 15 min. The supernatantwas removed by aspiration and the radioactivity associated with thepellet was counted in a gamma counter.

Monoclonal antibodies 4.1.3, 5.2.3, 4.8, 5.12.14, and 12.3.9 inhibitedgreater than 85% of the binding of IL-8 to human neutrophils at a 1:25molar ratio of IL-8 to mAb. On the other hand, monoclonal antibodies9.2.4 and 8.9.1 appeared to enhance the binding of IL-8 to its receptorson human neutrophils. Since a control mouse IgG also enhanced thebinding of IL-8 on neutrophils, the enhancement of IL-8 binding to itsreceptors by mAb 9.2.4 and 8.9.1 appears to be nonspecific. Thus,monoclonal antibodies, 4.1.3, 5.1.3, 4.8, 5.12.14, and 12.3.9 arepotential neutralizing monoclonal antibodies while monoclonal antibodies8.9.1 and 9.2.4 are non-neutralizing monoclonal antibodies.

The ability of the anti-IL-8 antibodies to block neutrophil chemotaxisinduced by IL-8 was tested as follows. Neutrophil chemotaxis induced byIL-8 was determined using a Boyden chamber method (Larsen, et al.Science 243:1464 (1989)). One hundred μl of human neutrophils (10⁶cells/ml) resuspended in RPMI containing 0.1% BSA were placed in theupper chamber and 29 μl of the IL-8 (20 nM) with or without monoclonalantibodies were placed in the lower chamber. Cells were, incubated for 1hr at 37° C. Neutrophils migrated into the lower chamber were stainedwith Wright's Giemsa stain and counted under the microscope (100×magnification). Approximately 10 different fields per experimental groupwere examined. Neutralizing monoclonal antibodies 5.12.14 and 4.1.3blocked almost 70% of the neutrophil chemotactic activity of IL-8 at1:10 ratio of IL-8 to mAb.

The isoelectric focusing (IEF) pattern of each mAb was determined byapplying purified antibodies on an IEF polyacrylamide gel (pH 3–9,Pharmacia) using the Fast gel system (Pharmacia, Piscataway, N.J.). TheIEF gel was pretreated with pharmalyte containing 1% Triton X100 (Sigma,St. Louis, Mo.) for 10 min before loading the samples. The IEF patternwas visualized by silver staining according to the instructions from themanufacturer. All of the monoclonal antibodies had different IEFpatterns, confirming that they originated from different clones. The pIvalues for the antibodies are listed in Table 3.

All these monoclonal antibodies bound equally well to both (ala-IL-8)₇₇and (ser-IL-8)₇₂ forms of IL-8. Because IL-8 has greater than 30%sequence homology with certain other members of the platelet factor 4(PF4) family of inflammatory cytokines such as β-TG (Van Damme et al.,Eur. J. Biochem. 181:337(1989); Tanaka et al., FEB 236(2):467 (1988))and PF4 (Deuel et al., Proc. Natl. Acad. Sci. U.S.A. 74:2256 (1977)),they were tested for possible cross reactivity to β-TG and PF4, as wellas to another neutrophil activating factor, C5a. No detectable bindingto any of these proteins was observed, with the exception of mAb 4.1.3,which had a slight cross reactivity to β-TG.

One of the antibodies, mAb 5.12.14, was further studied to determinewhether it could block the IL-8 mediated release of elastase byneutrophils. Briefly, human neutrophils were resuspended in Hanksbalanced salt solution (Gibco, Grand Island, N.Y.) containing 1.0% BSA,Fraction V (Sigma, St. Louis, Mo.), 2 mg/ml alpha-D-glucose (Sigma), 4.2mM sodium bicarbonate (Sigma) and 0.01 M HEPES, pH 7.1 (JRH Bioscience,Lenexa, Kans.). A stock of cytochalasin B (Sigma) was prepared (5 mg/mlin dimethylsulfoxide (Sigma) and stored at 2–8° C. Cytochalasin B wasadded to the neutrophil preparation to produce a final concentration of5 μg/ml, and incubated for 15 min at 37° C. Human IL-8 was incubatedwith mAb 5.12.14 (20 μl), or a negative control antibody, in 1 mlpolypropylene tubes (DBM Scientific, San Fernando, Calif.) for 30 min at37° C. The final assay concentrations of IL-8 were 50 and 500 nM. Themonoclonal antibodies were diluted to produce the following ratios(IL-8:Mab): 1:50, 1:10, 1:2, 1:1, and 1:0.25. Cytochalasin B-treatedneutrophils were added (100 μl/tube) and incubated for 2 hours at 25° C.The tubes were centrifuged (210×g, 2–8° C.) for 10 min, and supernatantswere transferred to 96 well tissue culture plates (30 μl/well). Elastasesubstrate stock, 10 mMmethoxysuccinyl-alanyl-alanyl-propyl-valyl-p-nitroanilide (Calbiochem,La Jolla, Calif.) in DMSO was prepared and stored at 2–8° C. Elastasesubstrate solution (1.2 mM substrate, 1.2 M NaCl (Mallinckrodt, Paris,Ky.), 0.12 M HEPES pH 7.2 in distilled water) was added (170 μl/well) tothe supernatants and incubated for 0.5 to 2 hours at 37° C. (untilcontrol O.D. of 1.0 was reached). Absorbance was measured at 405 nm (SLT340 ATTC plate reader, SLT Lab Instruments, Austria).

The results are shown in FIG. 1. At a 1:1 ratio of IL-8 to mAb 5.12.14,the antibody was able to effectively block the release of elastase fromneutrophils.

The hybridoma producing antibody 5.12.14 was deposited on Feb. 15, 1993with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., U.S.A. (ATCC) and assigned ATTC Accession No. HB 11553.

B. Generation and Characterization of Monoclonal Antibodies AgainstRabbit IL-8

Antibodies against rabbit IL-8 were generated in essentially the sameprocess as anti-human IL-8 antibodies using rabbit IL-8 as immunogen(kindly provided by C. Broaddus; see also Yoshimura et al. J. Immunol.146:3483 (1991)). The antibody was characterized as described above forbinding to other cytokines coated onto ELISA plates; no measurablebinding was found to MGSA, fMLP, C5a, b-TG, TNF, PF4, or IL-1.

The hybridoma producing antibody 6G4.2.5 was deposited on Sep. 28, 1994,with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., U.S.A. (ATCC) and assigned ATTC Accession No. HB 11722.

Recombinant human-murine chimeric Fabs for 5.12.14 and 6G4.2.5 wereconstructed as described below. A chimeric 6G.4.25 Fab is compared witha chimeric 5.12.14 Fab in detail below.

1. Inhibition of IL-8 Binding to Human Neutrophils by 5.12.14-Fab and6G4 2.5-Fab

The ability of the two chimeric Fabs, 5.12.14-Fab and 6G4.2.5-Fab, toefficiently bind IL-8 and prevent IL-8 from binding to IL-8 receptors onhuman neutrophils was determined by performing a competition bindingassay which allows the calculation of the IC₅₀—concentration required toachieve 50% inhibition of IL-8 binding.

Human neutrophils (5×10⁵) were incubated for 1 hour at 4° C. with 0.5 nM¹²⁵I-IL-8 in the presence of various concentrations (0 to 300 nM) of5.12.14-Fab, 6G4.2.5-Fab, an isotype control (4D5-Fab) or unlabeledIL-8. After the incubation, the unbound ¹²⁵I-IL-8 was removed bycentrifugation through a solution of 20% sucrose and 0.1% bovine serumalbumin in phosphate buffered saline and the amount of ¹²⁵I-IL-8 boundto the cells was determined by counting the cell pellets in a gammacounter. FIG. 2 demonstrates the inhibition of ¹²⁵I-IL-8 binding toneutrophils by unlabeled IL-8. FIG. 3 demonstrates that a negativeisotype matched Fab does not inhibit the binding of ¹²⁵I-IL-8 to humanneutrophils. Both the anti-IL-8 Fabs, 5.12.14 Fab (FIG. 4) and 6G.4.25Fab (FIG. 5) were able to inhibit the binding of ¹²⁵I-IL-8 to humanneutrophils with an average IC₅₀ of 1.6 nM and 7.5 nM, respectively.

2. Inhibition of IL-8-Mediated Neutrophil Chemotaxis by 5.12.14-Fab and6G4.2.5-Fab

Human neutrophils were isolated, counted and resuspended at 5×10⁶cells/ml in Hank's balanced salt solution (abbreviated HBSS; withoutcalcium and magnesium) with 0.1% bovine serum albumin. The neutrophilswere labeled by adding calcein AM (Molecular Probe, Eugene, Oreg.) at afinal concentration of 2.0 μM. Following a 30 minute incubation at 37°C., cells were washed twice with HBSS-BSA and resuspended at 5×10⁶cells/ml.

Chemotaxis experiments were carried out in a Neuro Probe (Cabin John,Md.) 96-well chamber, model MBB96. Experimental samples (buffer onlycontrol, IL-8 alone or IL-8+Fabs) were loaded in a Polyfiltronics96-well View plate (Neuro Probe Inc.) placed in the lower chamber. 100μl of the calcein AM-labeled neutrophils were added to the upperchambers and allowed to migrate through a 5 micrometer porosity PVP freepolycarbonate framed filter (Neuro Probe Inc.) toward the bottom chambersample. The chemotaxis apparatus was then incubated for 40 to 60 minutesat 37° C. with 5% CO₂. At the end of the incubation, neutrophilsremaining in the upper chamber were aspirated and upper chambers werewashed three times with PBS. Then the polycarbonate filter was removed,non-migrating cells were wiped off with a squeegee wetted with PBS, andthe filter was air dried for 15 minutes.

The relative number of neutrophils migrating through the filter(Neutrophil migration index) was determined by measuring fluorescenceintensity of the filter and the fluorescence intensity of the contentsof the lower chamber and adding the two values together. Fluorescenceintensity was measured with a CytoFluor 2300 fluorescent plate reader(Millipore Corp. Bedford, Mass.) configured to read a Corning 96-wellplate using the 485–20 nm excitation filter and a 530-25 emissionfilter, with the sensitivity set at 3.

The results are shown in FIGS. 6 and 7. FIG. 6 demonstrates theinhibition of human IL-8 mediated neutrophil chemotaxis by chimeric6G4.2.5 and 5.12.14 Fabs. FIG. 7 demonstrates the relative abilities ofchimeric 6G4.2.5 and 5.12.14 Fabs to inhibit rabbit IL-8 mediatedneutrophil chemotaxis.

3. Inhibition of IL-8-Mediated Neutrophil Elastase Release by VariousConcentrations of 6G4.2.5 and 5.12.14 Fabs

Blood was drawn from healthy male donors into heparinized syringes.Neutrophils were isolated by dextran sedimentation, centrifugation overLymphocyte Separation Medium (Organon Teknika, Durham, N.C.), andhypotonic lysis of contaminating red blood cells as described by Bermanet al. (J. Cell Biochem. 52:183 (1993)). The final neutrophil pellet wassuspended at a concentration of 1×10⁷ cells/ml in assay buffer, whichconsisted of Hanks Balanced Salt Solution (GIBCO, Grand Island, N.Y.)supplemented with 1.0% BSA (fraction V, Sigma. St. Louis, Mo.), 2 mg/mlglucose, 4.2 mM sodium bicarbonate, and 0.01 M HEPES, pH 7.2. Theneutrophils were stored at 4° C. for not longer than 1 hr.

IL-8 (10 μl) was mixed with anti-IL-8 Fab, an isotype control Fab, orbuffer (20 μl) in 1 ml polypropylene tubes and incubated in a 37° C.water bath for 30 min. IL-8 was used at final concentrations rangingfrom 0.01 to 1000 nM in dose response studies (FIG. 8) and at a finalconcentration of 100 nM in the experiments addressing the effects of theFabs on elastase release (FIGS. 9 and 10). Fab concentrations rangedfrom approximately 20 nM to 300 nM, resulting in Fab:IL-8 molar ratiosof 0.2:1 to 3:1. Cytochalasin B (Sigma) was added to the neutrophilsuspension at a concentration of 5 μg/ml (using a 5 mg/ml stock solutionmade up in DMSO), and the cells were incubated for 15 min in a 37° C.water bath. Cytochalasin B-treated neutrophils (100 μl) were then addedto the IL-8/Fab mixtures. After a 3 hr incubation at room temperature,the neutrophils were pelleted by centrifugation (200×g for 5 min), andaliquots of the cell-free supernatants were transferred to 96 wellplates (30 μl/well). The elastase substrate,methoxysuccinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (Calbiochem,La Jolla, Calif.), was prepared as a 10 mM stock solution in DMSO andstored at 4° C. Elastase substrate working solution was prepared justprior to use (1.2 mM elastase substrate, 1.2 M NaCl, 0.12 M HEPES, pH7.2), and 170 μl was added to each sample-containing well. The plateswere placed in a 37° C. tissue culture incubator for 30 min or until anoptical density reading for the positive controls reached at least 1.0.Absorbance was measured at 405 nm using an SLT 340 plate reader (SLT LabInstruments, Austria).

FIG. 9 demonstrates the ability of the chimeric anti-IL-8 Fabs toinhibit elastase release from human neutrophils stimulated by humanIL-8; FIG. 10 demonstrates the relative abilities of the chimericanti-IL-8 Fabs to inhibit elastase release from human neutrophilsstimulated by rabbit IL-8.

C. Molecular Cloning of the Variable Light and Heavy Regions of theMurine 5.12.14 (Anti-IL-8) Monoclonal Antibody

Total RNA was isolated from 1×10⁸ cells (hybridoma cell line ATCCHB-11722) using the procedure described by Chomczynski and Sacchi (Anal.Biochem. 162:156 (1987)). First strand cDNA was synthesized byspecifically priming the mRNA with synthetic DNA oligonucleotidesdesigned to hybridize with regions of the murine RNA encoding theconstant region of the kappa light chain or the IgG2a heavy chain (theDNA sequence of these regions are published in Sequences of Proteins ofImmunological Interest, Kabat, E. A. et al. (1991) NIH Publication91–3242, V1–3.). Three primers (SEQ ID NOS: 1–6) were designed for eachof the light and heavy chains to increase the chances of primerhybridization and efficiency of first strand cDNA synthesis (FIG. 13).Amplification of the first strand cDNA to double-stranded (ds) DNA wasaccomplished using two sets of synthetic DNA oligonucleotide primers:one forward primer (SEQ ID NOS: 7–9) and one reverse primer (SEQ ID NO:10) for the light chain variable region amplification (FIG. 14) and oneforward primer (SEQ ID NOS: 11–14) and one reverse primer (SEQ ID NOS:11, 15, 14 and 13) for the heavy chain variable region amplification(FIG. 15). The N-terminal sequence of the first eight amino acids ofeither the light or heavy chains of 5.12.14 was used to generate aputative murine DNA sequence corresponding to this region. (A total of29 amino acids was sequenced from the N-terminus of both the light chainand heavy chain variable regions using the Edman degradation proteinsequencing technique.) This information was used to design the forwardamplification primers which were made degenerate in the third positionfor some codons to increase the chances of primer hybridization to thenatural murine DNA codons and also included the unique restriction site,MluI, for both the light chain variable region forward primer and theheavy chain variable region forward primer to facilitate ligation to the3′ end of the STII element in the cloning vector. The reverseamplification primers were designed to anneal with the murine DNAsequence corresponding to a portion of the constant region of the lightor heavy chains near the variable/constant junction. The light chainvariable region reverse primer contained a unique BstBI restriction siteand the heavy chain variable region reverse primer contained a uniqueApaI restriction site for ligation to the 5′ end of either the humanIgG1 constant light or IgG1 constant heavy regions in the vectors,pB13.1 (light chain) and pB14 (heavy chain). The polymerase chainreaction using these primer sets yielded DNA fragments of approximately400 bp. The cDNA encoding the 5.12.14 light chain variable region wascloned into the vector pB13.1, to form pA51214VLand the 5.12.14 heavychain variable region was cloned into the vector, pB14, to formpA51214VH. The cDNA inserts were characterized by DNA sequencing and arepresented in the DNA sequence (SEQ ID NO: 16) and amino acid sequence(SEQ ID NO: 1.7) of FIG. 16 (murine light chain variable region) and inthe DNA sequence (SEQ ID NO: 18) and amino acid (SEQ ID NO: 19) of FIG.17 (murine heavy chain variable region).

D. Construction of a 5.12.14 Fab Vector

In the initial construct, pA51214VL, the amino acids between the end ofthe 5.12.14 murine light chain variable sequence and the unique cloningsite, BstBI, in the human IgG1 constant light sequence were of murineorigin corresponding to the first 13 amino acids of the murine IgG1constant region (FIG. 16). Therefore, this plasmid contained asuperfluous portion of the murine constant region separating the 5.12.14murine light chain variable region and the human light chain IgG1constant region. This intervening sequence would alter the amino acidsequence of the chimera and most likely produce an incorrectly foldedFab. This problem was addressed by immediately truncating the cDNA cloneafter A 109 and re-positioning the BstBI site to the variable/constantjunction by the polymerase chain reaction. FIG. 18 shows theamplification primers used to make these modifications. The forwardprimer, VL.front (SEQ ID NO: 20), was designed to match the last fiveamino acids of the STII signal sequence, including the MluI cloningsite, and the first 4 amino acids of the 5.12.14 murine light chainvariable sequence. The sequence was altered from the original cDNA inthe third position of the first two codons D1 (T to C) and I2 (C to T)to create a unique EcoRV cloning site which was used for laterconstructions. The reverse primer, VL.rear (SEQ ID NO: 21), was designedto match the first three amino acids of the human IgG1 constant lightsequence and the last seven amino acids of the 5.12.14 light chainvariable sequence which included a unique BstBI cloning site. In theprocess of adding the BstBI site, the nucleotide sequence encodingseveral amino acids were altered: L106 (TTG to CTT), K107 (AAA to CGA)resulting in a conservative amino acid substitution to arginine, andR108 (CGG to AGA). The PCR product encoding the modified 5.12.14 lightchain variable sequence was then subcloned into pB13.1 in a two-partligation. The MluI-BstBI digested 5.12.14 PCR product encoding the lightchain variable region was ligated into MluI-BstBI digested vector toform the plasmid, pA51214VL′. The modified cDNA was characterized by DNAsequencing. The coding sequence for the 5.12.14 light chain is shown inFIG. 19.

Likewise, the DNA sequence between the end of the heavy chain variableregion and the unique cloning site, ApaI, in the human IgG1 heavy chainconstant domain of pA51214VH was reconstructed to change the amino acidsin this area from murine to human. This was done by the polymerase chainreaction. Amplification of the murine 5.12.14 heavy chain variablesequence was accomplished using the primers shown in FIG. 18. Theforward PCR primer (SEQ ID NO: 22) was designed to match nucleotides867–887 in pA51214VH upstream of the STII signal sequence and theputative cDNA sequence encoding the heavy chain variable region andincluded the unique cloning site SpeI. The reverse PCR primer (SEQ IDNO: 23) was designed to match the last four amino acids of the 5.12.14heavy chain variable sequence and the first six amino acidscorresponding to the human IgG1 heavy constant sequence which alsoincluded the unique cloning site, ApaI. The PCR product encoding themodified 5.12.14 heavy chain variable sequence was then subcloned to theexpression plasmid, pMHM24.2.28 in a two-part ligation. The vector wasdigested with SpeI-ApaI and the SpeI-ApaI digested 5.12.14 PCR productencoding the heavy chain variable region was ligated into it to form theplasmid, pA51214VH′. The modified cDNA was characterized by DNAsequencing. The coding sequence for the 5.12.14 heavy chain is shown inthe DNA sequence (SEQ ID NO: 26) and amino acid sequence (SEQ ID NO: 27)of FIGS. 20A-20B.

The first expression plasmid, pantiIL-8.1, encoding the chimeric Fab of5.12.14 was made by digesting pA51214VH′ with EcoRV and Bpu1102I toreplace the EcoRV-Bpu1102I fragment with a EcoRV-Bpu1102I fragmentencoding the murine 5.12.14 light chain variable region of pA51214VL′.The resultant plasmid thus contained the murine-human variable/constantregions of both the light and heavy chains of 5.12.14.

Preliminary analysis of Fab expression using pantiIL-8.1 showed that thelight and heavy chains were produced intracellularly but very little wasbeing secreted into the periplasmic space of E. coli. To correct thisproblem, a second expression plasmid was constructed.

The second expression plasmid, pantiIL-8.2, was constructed using theplasmid, pmy187, as the vector. Plasmid pantiIL-8.2 was made bydigesting pmy187 with MluI and SphI and the MluI (partial)-SphI fragmentencoding the murine 5.12.14 murine-human chimeric Fab of pantiIL-8.1 wasligated into it. The resultant plasmid thus contained the murine-humanvariable/constant regions of both the light and heavy chains of 5.12.14.

The plasmid pantiIL-8.2 was deposited on Feb. 10, 1995 with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., U.S.A.(ATCC) and assigned ATTC Accession No. ATCC 97056.

E. Molecular Cloning of the Variable Light and Heavy Regions of theMurine 6G4.2.5 Monoclonal Antibody

Total RNA was isolated from 1×10⁸ cells (hybridoma cell line 6G4.2.5)using the procedure described by Chomczynski and Sacchi (Anal. Biochem.162:156 (1987)). First strand cDNA was synthesized by specificallypriming the mRNA with synthetic DNA oligonucleotides designed tohybridize with regions of the murine RNA encoding the constant region ofthe kappa light chain or the IgG2a heavy chain (the DNA sequence ofthese regions are published in Sequences of Proteins of ImmunologicalInterest, Kabat et al. (1991) NIH Publication 91–3242, V1–3). Threeprimers (SEQ ID NOS: SEQ ID NOS: 1–6) were designed for each the lightand heavy chains to increase the chances of primer hybridization andefficiency of first strand cDNA synthesis (FIG. 21). Amplification ofthe first strand cDNA to double-stranded (ds) DNA was accomplished usingtwo sets of synthetic DNA oligonucleotide primers: one forward primer(SEQ ID NOS: 28–30) and one reverse primer (SEQ ID NO: 31) for the lightchain variable region amplification (FIG. 22) and one forward primer(SEQ ID NOS: 32–33) and one reverse primer (SEQ ID NOS: 11, 15, 14 and13) for the heavy chain variable region amplification (FIG. 23). TheN-terminal sequence of the first eight amino acids of either the lightor heavy chains of 6G4.2.5 was used to generate a putative murine DNAsequence corresponding to this region. (A total of 29 amino acids weresequenced from the N-terminus of both the light chain and heavy chainvariable regions using the Edman degradation protein sequencingtechnique.) This information was used to design the forwardamplification primers which were made degenerate in the third positionfor some codons to increase the chances of primer hybridization to thenatural murine DNA codons and also included the unique restriction site,NsiI, for the light chain variable region forward primer and the uniquerestriction site, MluI, for the heavy chain variable region forwardprimer to facilitate ligation to the 3′ end of the STII element in thevector, pchimFab. The reverse amplification primers were designed toanneal with the murine DNA sequence corresponding to a portion of theconstant region of the light or heavy chains near the variable/constantjunction. The light chain variable region reverse primer contained aunique MunI restriction site and the heavy chain variable region reverseprimer contained a unique ApaI restriction site for ligation to the 5′end of either the human IgG1 constant light or IgG1 constant heavyregions in the vector, pchimFab. The polymerase chain reaction usingthese primer sets yielded DNA fragments of approximately 400 bp and werecloned individually into the vector, pchimFab, to form p6G425VL andp6G425VH. The cDNA inserts were characterized by DNA sequencing and arepresented in the DNA sequence (SEQ ID NO: 34) and amino acid sequence(SEQ ID NO: 35) of FIG. 24 (murine light chain variable region) and theDNA sequence (SEQ ID NO: 36) and amino acid sequence (SEQ ID NO: 37) ofFIG. 25 (murine heavy chain variable region).

F. Construction of a 6G4.2.5 Chimeric Fab Vector

In the initial construct, p6G425VL, the amino acids between the end ofthe 6G4.2.5 murine light chain variable sequence and the unique cloningsite, MunI, in the human IgG1 constant light sequence were of murineorigin. These amino acids must match the human IgG1 amino acid sequenceto allow proper folding of the chimeric Fab. Two murine amino acids,D115 and S121, differed dramatically from the amino acids found in theloops of the β-strands of the human IgG1 constant domain and wereconverted to the proper human amino acid residues, V115 and F121, bysite-directed mutagenesis using the primers (SEQ ID NOS: 38, 39, 40)shown in FIG. 26. These specific mutations were confirmed by DNAsequencing and the modified plasmid named p6G425VL′. The coding sequenceis shown in the DNA sequence (SEQ ID NO: 41) and amino acid sequence(SEQ ID NO: 42) of FIGS. 27A–27B.

Likewise, the DNA sequence between the end of the heavy chain variableregion and the unique cloning site, ApaI, in the human IgG1 heavy chainconstant domain of p6G425VH was reconstructed to change the amino acidsin this area from murine to human. This process was facilitated by thediscovery of a BstEII site near the end of the heavy chain variableregion. This site and the ApaI site were used for the addition of asynthetic piece of DNA encoding the corresponding IgG human amino acidsequence. The synthetic oligo-nucleotides shown in FIG. 26 were designedas complements of one another to allow the formation of a 0.27 bp pieceof ds DNA. The construction was performed as a three-part ligationbecause the plasmid, p6G425VH, contained an additional BstEI site withinthe vector sequence. A 5309 bp fragment of p6G425VH digested withMluI-ApaI was ligated to a 388 bp fragment carrying the 6G4.2.5 heavychain variable region and a 27 bp synthetic DNA fragment encoding thefirst six amino acids of the human IgG1 constant region to form theplasmid, p6G425VH′. The insertion of the synthetic piece of DNA wasconfirmed by DNA sequencing. The coding sequence is shown in the DNAsequence (SEQ ID NO: 43) and amino acid sequence (SEQ ID NO: 44) ofFIGS. 28A–28B.

The expression plasmid, p6G425chim2, encoding the chimeric Fab of6G4.2.5 was made by digesting p6G425chimVL′ with MluI and ApaI to removethe STII-murine HPC4 heavy chain variable region and replacing it withthe MluI-ApaI fragment encoding the STII-murine 6G4.2.5 heavy chainvariable region of p6G425chimVH′. The resultant plasmid thus containedthe murine-human variable/constant regions of both the light and heavychains of 6G4.2.5.

The plasmid p6G425chim2 was deposited on Feb. 10, 1995 with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., U.S.A.(ATCC) and assigned ATTC Accession No. 97055.

G. Construction of Humanized Versions of Anti-IL-8 Antibody 6G4.2.5

The murine cDNA sequence information obtained from the hybridoma cellline, 6G4.2.5, was used to construct recombinant humanized variants ofthe murine anti-IL-8 antibody. The first humanized variant, F(ab)-1, wasmade by grafting synthetic DNA oligonucleotide primers encoding themurine CDRs of the heavy and light chains onto a phagemid vector, pEMX1(Werther et al., J. Immunol, 157: 4986–4995 (1996)), which contains ahuman 6-subgroup I light chain and a human IgG1 subgroup III heavy chain(FIG. 29). Amino acids comprising the framework of the antibody thatwere potentially important for maintaining the conformations necessaryfor high affinity binding to IL-8 by the complementarity-determiningregions (CDR) were identified by comparing molecular models of themurine and humanized 6G4.2.5 (F(ab)-1) variable domains using methodsdescribed by Carter et al., PNAS 89:4285 (1992) and Eigenbrot, et. al.,J. Mol. Biol. 229:969 (1993). Additional humanized framework variants(F(ab) 2–9) were constructed from the information obtained from thesemodels and are presented in Table 2 below. In these variants, thesite-directed mutagenesis methods of Kunkel, Proc. Natl. Acad. Sci USA),82:488 (1985) were utilized to exchange specific human frameworkresidues with their corresponding 6G4.2.5 murine counterparts.Subsequently, the entire coding sequence of each variant was confirmedby DNA sequencing. Expression and purification of each F(ab) variant wasperformed as previously described by Werther et. al., supra, with theexception that hen egg white lysozyme was omitted from the purificationprotocol. The variant antibodies were analyzed by SDS-PAGE, electrospraymass spectroscopy and amino acid analysis.

TABLE 4 Humanized 6G425 Variants IC50^(c) Variant Version TemplateChanges^(a) Purpose^(b) Mean S.D. N F(ab)-1 version 1 CDR Swap 63.0 12.34 F(ab)-2 version 2 F(ab)-1 PheH67

packaging w/ CDR H2 106.0 17.0 2 F(ab)-3 version 3 F(ab)-1 ArgH71

packaging w/ CDRs H1, H2 79.8 42.2 4 F(ab)-4 version 6 F(ab)-1 IleH69

packaging w/ CDR H2 44.7 9.0 3 F(ab)-5 version 7 F(ab)-1 LeuH78

packaging w/ CDRs H1, H2 52.7 31.0 9 F(ab)-6 version 8 F(ab)-1 IleH69

combine F(ab)-4 and -5 34.6 6.7 7 LeuH78

F(ab)-7 version 16 F(ab)-6 LeuH80

packaging w/ CDR H1 38.4 9.1 2 F(ab)-8 version 19 F(ab)-6 ArgH38

packaging w/ CDR H2 14.0 5.7 2 F(ab)-9 version 11 F(ab)-6 GluH6

packaging w/ CDR H3 19.0 5.1 7 Chimeric^(d) 11.4 7.0 1 F(ab) 3rhu4D5^(e) >200 μM 5 F(ab) ^(a)Amino acid changes made relative to thetemplate used. Murine residues are in bold italics and residue numberingis according to Kabat et al. ^(b)Purpose for making changes based uponinteractions observed in molecular models of the humanized and murinevariable domains. ^(c)nM concentration of variant necessary to inhibitbinding of iodinated IL-8 to human neutrophils in the competitivebinding assay. ^(d)Chimeric F(ab) is a (F(ab) which carries the murineheavy and light chain variable domains fused to the human light chain kIconstant domain and the human heavy chain subgroup III constant domain Irespectively. ^(e)rhu4D5F(ab) is of the same isotype as the humanized6G425 F(ab)s and is a humanized anti-HER2 F(ab) and therefore should notbind to IL8.

The first humanized variant, F(ab)-1, was an unaltered CDR swap in whichall the murine CDR amino acids defined by both x-ray crystallography andsequence hypervariability were transferred to the human framework. Whenthe purified F(ab) was tested for its ability to inhibit ¹²⁵I-IL-8binding to human neutrophils according to the methods described inSection (B)(1) above, a 5.5 fold reduction in binding affinity wasevident as shown in Table 4 above. Subsequent versions of F(ab)-1 wereengineered to fashion the 3-dimensional structure of the CDR loops intoa more favorable conformation for binding IL-8. The relative affinitiesof the F(ab) variants determined from competition binding experimentsusing human neutrophils as described in Section (B)(1) above arepresented in Table 4 above. A slight decrease in IL-8 binding (<2 fold)was observed for F(ab)-2–3 while only slight increases in IL-8 bindingwere noted for F(ab)3–5. Variant F(ab)-6 had the highest increase inaffinity for IL-8 (approximately 2 fold), exhibiting an IL-8 bindingaffinity of 34.6 nM compared to the F(ab)-1 IL-8 binding affinity of 63nM. The substitutions of murine Leu for Ile at H69 and murine Ala forLeu at H78 are predicted to influence the packing of CDRs H1 and H2.Further framework substitutions using the F(ab)-6 variant as templatewere made to bring the binding affinity closer to that of the chimericF(ab). In-vitro binding experiments revealed no change in affinity forF(ab)-7 (38.4 nM) but a significant improvement in affinity forF(ab)-8/9 of 14 nM and 19 nM, respectively. By analysis of a 3-Dcomputer-generated model of the anti-IL-8 antibody, it was hypothesizedthat the substitution of murine Lys for Arg at H38 in F(ab)-8 influencesCDR-H2 while a change at H6 of murine Gln for Glu in F(ab)-9 affectsCDR-H3. Examination of the human antibody sequences with respect toamino acid variability revealed that the frequency of Arg at residue H38is >99% whereas residue H6 is either Gln ˜20% or Glu ˜80% (Kabat et.al., Sequences of Proteins of Immunological Interest 5th Ed. (1991)).Therefore, to reduce the likelihood of causing an immune response to theantibody, F(ab)-9 was chosen over F(ab)-8 for further affinitymaturation studies. Variant F(ab)-9 was also tested for its ability toinhibit IL-8-mediated chemotaxis (FIG. 30). This antibody was able toblock neutrophil migration induced by wild-type human IL-8, humanmonomeric IL-8 and Rhesus IL-8 with IC₅₀=s of approximately 12 nM, 15nM, and 22 nM, respectively, in IL-8 mediated neutrophil chemotaxisinhibition assays performed as described in Section (B)(2) above. Theamino acid sequence for variant F(ab)-8 is provided in FIG. 31 c. TheF(ab)-8 was found to block human and rhesus IL-8-mediated chemotaxiswith IC₅₀=s of 12 nM and 10 nM, respectively, in IL-8 mediatedneutrophil chemotaxis inhibition assays performed as described inSection (B)(2) above.

H. Construction of an Anti-IL-8-Gene III Fusion Protein for PhageDisplay and Alanine Scanning Mutagenesis

An expression plasmid, pPh6G4.V11, encoding a fusion protein (heavychain of the humanized 6G4.2.5 version 11 antibody and the M13 phagegene-III coat protein) and the light chain of the humanized 6G4.2.5version 11 antibody was assembled to produce a monovalent display of theanti-IL-8 antibody on phage particles. The construct was made bydigesting the plasmid, pFPHX, with EcoRV and ApaI to remove the existingirrelevant antibody coding sequence and replacing it with a 1305 bpEcoRV-ApaI fragment from the plasmid, p6G4.V11, encoding the humanized6G4.2.5 version 11 anti-IL-8 antibody. The translated sequence of thehumanized 6G4.2.5 version 111 heavy chain (SEQ ID NO: 52), peptidelinker and gene III coat protein (SEQ ID NO: 53) is shown in FIG. 31A.The pFPHX plasmid is a derivative of phGHam-3 which contains an in-frameamber codon (TAG) between the human growth hormone and gene-III DNAcoding sequences. When transformed into an amber suppressor strain of E.coli, the codon (TAG) is read as Glutamate producing a growth hormone(hGH)-gene III fusion protein. Likewise, in a normal strain of E. coli,the codon (TAG) is read as a stop preventing translational read-throughinto the gene-III sequence and thus allowing the production of solublehGH. The pGHam-3 plasmid is described in Methods: A Companion to Methodsin Enzymology, 3:205 (1991). The final product, pPh6G4.V11, was used asthe template for the alanine scanning mutagenesis of the CDRs and forthe construction of randomized CDR libraries of the humanized 6G4.V11antibody.

I. Alanine Scanning Mutagenesis of Humanized Antibody 6G4.2.5 Version 11

The solvent exposed amino acid residues in the CDRs of the humanizedanti-IL-8 6G4.2.5 version 11 antibody (h6G4V11) were identified byanalysis of a 3-D computer-generated model of the anti-IL-8 antibody. Inorder to determine which solvent exposed amino acids in the CDRs affectbinding to interleukin-8, each of the solvent exposed amino acids wasindividually changed to alanine, creating a panel of mutant antibodieswherein each mutant contained an alanine substitution at a singlesolvent exposed residue. The alanine scanning mutagenesis was performedas described by Leong et. al., J. Biol. Chem., 269: 19343 (1994)).

The IC₅₀'s (relative affinities) of h6G4V11 wt and mutated antibodieswere established using a Competition Phage ELISA Assay described byCunningham et. al., (EMBO J. 13:2508 (1994)) and Lee et. al., (Science270:1657 (1995)). The assay measures the ability of each antibody tobind IL-8 coated onto a 96-well plate in the presence of variousconcentrations of free IL-8 (0.2 to 1 uM) in solution. The first step ofthe assay requires that the concentrations of the phage carrying thewild type and mutated antibodies be normalized, allowing a comparison ofthe relative affinities of each antibody. The normalization wasaccomplished by titering the phage on the IL-8 coated plates andestablishing their EC₅₀. Sulfhydryl coated 96-well binding plates(Corning-Costar; Wilmington, Mass.) were incubated with a 0.1 mg/mlsolution of K64C IL-8 (Lysine 64 is substituted with Cysteine to allowthe formation of a disulfide bond between the free thiol group of K64CIL-8 and the sulfhydryl coated plate, which results in the positioningof the IL-8 receptor binding domains towards the solution interface) inphosphate buffered saline (PBS) pH 6.5 containing 1 mM EDTA for 1 hourat 25° C. followed by three washes with PBS and a final incubation witha solution of PBS containing 1.75 mg/ml of L-cysteine-HCl and 0.1MNaHCO₃ to block any free reactive sulfhydryl groups on the plate. Theplates were washed once more and stored covered at 4° C. with 200 ul ofPBS/well. Phage displaying either the reference antibody, h6G4V11, orthe mutant h6G4V11 antibodies were grown and harvested by PEGprecipitation. The phage were resuspended in 500 ul 10 mM Tris-HCl pH7.5, 1 mM EDTA and 100 mM NaCl and held at 4° C. for no longer than 3hours. An aliquot of each phage was diluted 4-fold in PBS containing0.05% Tween-20 (BioRad, Richmond, Calif.) and 0.5% BSA RIA grade (Sigma,St. Louis, Mo.) (PBB) and added to IL-8 coated plates blocked for atleast 2 hours at 25° C. with 50 mg/ml skim milk powder in 25 mMCarbonate Buffer pH 9.6. The phage were next serially diluted in 3 foldsteps down the plate from well A through H. The plates were incubatedfor 1 hour at 25° C. followed by nine quick washes with PBS containing0.05% Tween-20 (PBST). The plates were then incubated with a 1:3200dilution of rabbit anti-phage antibody and a 1:1600 dilution ofsecondary goat-anti-rabbit Fc HRP-conjugated antibody for 15 minutes at25° C. followed by nine quick washes with PBST. The plates weredeveloped with 80 ul/well of 1 mg/ml OPD (Sigma, St. Louis, Mo.) inCitrate Phosphate buffer pH 5.0 containing 0.015% H₂O₂ for 4 minutes at25° C. and the reaction stopped with the addition of 40 ul of 4.5MH₂SO₄. The plates were analyzed at wavelength 8492 in a SLT model340ATTC plate reader (SLT Lab Instruments). The individual EC₅₀=s weredetermined by analyzing the data using the program Kaleidagraph (SynergySoftware, Reading, Pa.) and a 4-parameter fit equation. The phage heldat 4° C. were then immediately diluted in PBB to achieve a finalconcentration corresponding to their respective EC₅₀ or target OD₄₉₂ forthe competition segment of the experiment, and dispensed into a 96 wellplate containing 4-fold serial dilutions of soluble IL-8 ranging from 1uM in well A and ending with 0.2 uM in well H. Using a 12-channel pipet,100 ul of the phage/IL-8 mixture was transferred to an IL-8 coated96-well plate and executed as described above. Each sample was done intriplicate—3 columns/sample.

TABLE 5 Relative Affinities (IC50) for Alanine-scan Anti-IL-8 6G4V11 CDRMutants CDR Amino Acid Residue Avg IC50 (nM) Std Dev V11 Reference 11.56.4 CDR-L1 S26 6.3 2.9 Q27 10.2 2.4 S28 14.2 5.2 V30 29.1 12.3 H31 580.3243.0 I33 64.2 14.6 N35 3.3 0.7 T36 138.0 nd Y37 NDB nd CDR-L2 K55 24.214.9 V56 15.5 3.8 S57 12.4 4.0 N58 17.6 3.7 R59 nd nd CDR-L3 S96 10.84.4 T97 70.6 55.2 H98 8.0 1.2 V99 19.6 1.9 CDR-H1 S28 8.6 3.1 S30 nd ndS31 7.8 2.5 H32 13.3 5.8 Y53 48.2 15.8 CDR-H2 Y50 35.6 13.0 D52 13.3 7.5S53 6.0 3.4 N54 96.0 5.8 E56 15.8 4.5 T57 8.4 1.6 T58 11.3 1.8 Y59 9.13.7 Q61 12.6 6.4 K64 18.5 12.1 CDR-H3 D96 NDB nd Y97 NDB nd R98 36.615.3 Y99 199.5 nd N100 278.3 169.4 D102 159.2 44 W103 NDB nd F104 NDB ndF105 209.4 72.3 D106 25.3 21.7 Each sample performed intriplicate/experiment. NDB = No Detectable Binding nd = value notdetermined* Residue numbering is according to Kabat et al.

The results of the alanine-scan are summarized in Table 5 above. Thealanine substitutions in of many of the mutant antibodies had little orno adverse effects (<3 fold) on the binding affinity for IL-8. Mutantsthat were found to exhibit no detectable binding of IL-8 (NDB)presumably contained disruptions in the conformational structure of theantibody conferred by crucial structural or buried amino acids in theCDR. Based on the results of the scan, CDR-H3 (heavy chain, 3rd CDR) wasidentified as the dominant binding epitope for binding IL-8. Alaninesubstitutions in this CDR resulted in a 3 to >26 fold decrease inbinding affinity. The amino acids, Y597, Y599 and D602 are of particularinterest because it was determined from the computer generated model ofthe anti-IL-8 antibody that these residues are solvent exposed and thatthese residues might participate in hydrogen bonding or chargeinteractions with IL-8 or other amino acids of the antibody thatinfluence either binding to IL-8 or the conformation of the CDR-H3 loopstructure. (See the model depicted in FIG. 32). Unexpected increases inbinding affinity (1.8>2.7 fold) were noted for S528 and S531 of CDR-H1and S553 of CDR-H2.

Surprisingly, a significant increase in binding affinity was observed inthe alanine mutant N35A located in CDR-L1 (light chain, 1st CDR). A 3–6fold increase in affinity was observed compared to the wild-type h6G4V11antibody. This augmentation of IL-8 binding could be the result of theclose proximity of N35A to CDR-H3. The alanine substitution may haveimparted a slight change in the conformation of CDR-L1 which alters thepacking interaction of neighboring amino acid residues on CDR-H3,thereby tweaking the loop of CDR-H3 into a conformation that facilitatesmore appropriate contacts with IL-8. Similarly, N35A may also influencethe orientation of amino acids in CDR-L1 or its interaction directlywith IL-8. Unexpected increases in affinity (˜2 fold) were also observedfor S26 of CDR-L1 and H98 of CDR-L3.

J. Characterization of Humanized Anti-IL-8 Antibody 6G4V11N35A

Soluble 6G4V11N35A Fab antibody was made by transforming an ambernon-suppressor strain of E. coli, 34B8, with pPh6G4.V11 and growing theculture in low phosphate medium for 24 hours. The periplasmic fractionwas collected and passed over a Hi-Trap Protein-G column (Pharmacia,Piscataway, N.J.) followed by a desalting and concentration step. Theprotein was analyzed by SDS-PAGE, mass spectrometry and amino acidanalysis. The protein had the correct size and amino acid composition(FIG. 35). The 6G4V11N35A Fab was tested for its ability to inhibit¹²⁵I-IL-8 binding to human neutrophils and to inhibit IL-8 mediatedneutrophil chemotaxis as described in Section (B)(1) and (B)(2) above.As shown in FIG. 33, hybridoma-derived intact murine antibody (6G4murine mAB), recombinant 6G4 murine-human chimera Fab, recombinanthumanized Fab versions 1 and 11, and 6G4V11N35A Fab were found toinhibit ¹²⁵I-IL-8 binding to human neutrophils with an average IC₅₀ of 5nM, 8 nM, 40 nM, 10 nM and 3 nM, respectively. The 6G4V11N35A Fab had atleast a 2-fold higher affinity than the 6G4.2.5 chimera Fab and a 3-foldhigher affinity than 6G4V11. As shown in FIG. 34, the 6G4V11N35A Fab wasfound to inhibit IL-8 mediated neutrophil chemotaxis induced by bothwild type and monomeric human IL-8, and by two different animal speciesof IL-8, namely, rabbit and rhesus. The irrelevant isotype control Fab(4D5) did not inhibit neutrophil migration. The average IC₅₀ values were3 nM (wt IL-8), 1 nM (monomeric IL-8), 5 nM (Rabbit IL-8), and 10 nM(Rhesus IL-8).

K. Construction of a 6G4V11N35A F(ab′)₂ Leucine Zipper

Production of a F(ab′)₂ version of the humanized anti-IL-8 6G4V11N35AFab was accomplished by constructing a fusion protein with the yeastGCN4 leucine zipper. The expression plasmid p6G4V11N35A.F(ab′)₂ was madeby digesting the plasmid p6G425chim2.fab2 with the restriction enzymesbsaI and apaI to remove the DNA sequence encoding the 6G4.2.5murine-human chimeric Fab and replacing it with a 2620 bp bsaI-apaIfragment from pPh6G4.V11N35A. The plasmid p6G425chim2.fab2 is aderivative of pS1130 which encodes a fusion protein (the GCN4 leucinezipper fused to the heavy chain of anti-CD18) and the light chain ofanti-CD18 antibody. The expression plasmid p6G4V11N35A.F(ab′)₂ wasdeposited on Feb. 20, 1996 with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md., U.S.A. (ATCC) and assigned ATCCAccession No. 97890. A pepsin cleavage site in the hinge region of theantibody facilitates the removal of the leucine zipper leaving the twoimmunoglobin monomers joined by the cysteines that generate theinterchain disulfide bonds. The DNA and protein sequence of theh6G4V11N35A.F(ab′)₂ are depicted in FIGS. 35–37.

An expression host cell was obtained by transforming E. coli strain 49D6with p6G4V11N35A.F(ab′)₂ essentially as described in Section (II)(3)(C)above. The transformed host E. coli 49D6 (p6G4V11N35A.F(ab′)₂) wasdeposited on Feb. 20, 1997 at the ATCC and assigned ATCC Accession No.98332. Transformed host cells were grown in culture, and the 6G4V11N35AF(ab′)₂ product was harvested from the host cell periplasmic spaceessentially as described in Section (II)(3)(F) above.

L. Characterization of the Humanized 6G4V11N35A F(ab′)₂ Leucine Zipper

The 6G4V11N35A Fab and F(ab′)₂ were tested for their ability to inhibit¹²⁵I-IL-8 binding to neutrophils according to the procedures describedin Section (B)(1) above. The displacement curves from a representativebinding experiment performed in duplicate is depicted in FIG. 38.Scatchard analysis of this data shows that 6G4V11N35A F(ab′)₂ inhibited¹²⁵I-IL-8 binding to human neutrophils with an average IC₅₀ of 0.7 nM(+/−0.2). This is at least a 7 fold increase in affinity compared to thehybridoma-derived intact murine antibody (average IC₅₀ of 5 nM) and atleast a 2.8 fold increase in affinity over the Fab version (average IC₅₀of 2 nM).

The 6G4V11N35A F(ab′)₂ was also tested for its ability to inhibit IL-8mediated neutrophil chemotaxis according to the procedures described inSection (B)(2) above. The results of a representative chemotaxisexperiment performed in quadruplicate are depicted in FIG. 39. As shownin FIG. 39, the 6G4V11N35A F(ab′)₂ inhibited human IL-8 mediatedneutrophil chemotaxis. The 6G4V11N35A F(ab′)₂ exhibited an average IC₅₀value of 1.5 nM versus 2.7 nM for the 6G4V11N35A Fab, which representsan approximately 2 fold improvement in the antibody's ability toneutralize the effects of IL-8. The irrelevant isotype control Fab (4D5)did not inhibit neutrophil migration. Furthermore, the 6G4V11N35AF(ab′)₂ antibody retained its ability to inhibit IL-8 mediatedneutrophil chemotaxis by monomeric IL-8 and by two different animalspecies of IL-8, namely rabbit and rhesus, in neutrophil chemotaxisexperiments conducted as described above. An individual experiment isshown in FIG. 40. The average IC₅₀ values were 1 nM (monomeric IL-8), 4nM (Rabbit IL-8), and 2.0 nM (Rhesus IL-8).

M. Random Mutagenesis of Light Chain Amino Acid (N35A) in CDR-L1 ofHumanized Antibody 6G4V11

A 3-fold improvement in the IC₅₀ for inhibiting ¹²⁵I-IL-8 binding tohuman neutrophils was observed when alanine was substituted forasparagine at position 35 in CDR-L1 (light chain) of the humanized6G4V11 mAb as described in Section (I) above. This result might beattributed to an improvement in the contact between the antigen-antibodybinding interfaces as a consequence of the replacement of a less bulkynonpolar side chain (R-group) that may have altered the conformation ofCDR-L1 or neighboring CDR-H3 (heavy chain) to become more accessible forantigen docking. The acceptance of alanine at position 35 of CDR-L1suggested that this position contributed to improved affinity and thatan assessment of the re-modeling of CDR loops/antigen-binding region(s)by other amino acids at this location was warranted. Selection of anaffinity matured version of the humanized 6G4.V11 mAB (Kunkel, T. A.,Proc. Natl. Acad. Sci. USA, 82:488 (1995)) was accomplished by randomlymutagenizing position 35 of CDR-L1 and constructing an antibody-phagelibrary. The codon or Asparagine (N) at position 35 of CDR-L1, wastargeted for randomization to any of the 20 known amino acids.

Initially, a stop template, pPh6G4.V11-stop, was made to eliminatecontaminating wild-type N35 sequence from the library. This wasaccomplished by performing site-directed mutagenesis (Muta-Gene Kit,Biorad, Ricmond, Calif.) of pPH6G4V11 (described in Section (H) above)to replace the codon (AAC) for N35 with a stop codon (TAA) using theprimer SL.97.2 (SEQ ID NO:63) (FIG. 42). The incorporation of the stopcodon was confirmed by DNA sequencing. Subsequently, uracil containingsingle-stranded DNA derived from E. coli CJ236 transformed with the stoptemplate was used to generate an antibody-phage library following themethod described by Lowman (Methods in Molecular Biology, 87 Chapter 25:1–15 (1997). The variants generated from this library were predicted toproduce a collection of antibodies containing one of the 20 known aminoacids at position N35 in CDR-L1. The amino acid substitutions wereaccomplished by site-directed mutagenesis using the degenerateoligonucleotide primer (SL.97.3) with the sequence NNS(N=A/G/T/C;S=G/C;) (SEQ ID NO: 64) (FIG. 42). This codon usage should allow for theexpression of any of the 20 amino acids—including the amber stop codon(TAG). The collection of antibody-phage variants was transfected into E.coli strain XL-1 blue (Stratagene, San Diego, Calif.) by electroporationand grown at 37° C. overnight to amplify the library. Selection of tightbinding humanized 6G4V11 Fab's were accomplished by panning the libraryon IL-8 coated 96-well plates as described in Section (I) above. Priorto panning, the number of phage/library was normalized to 1.1×10¹³phage/ml (which produces a maximum OD₂₇₀ reading=1 OD unit) and IL-8coated plates were incubated with blocking solution (25 mN Carbonatebuffer containing 50 mg/ml skim milk) for 2 hours before the addition ofphage (each sort used eight IL-8 coated wells/library). After theblocking and washing steps, every sort began with the addition of 100 ulof antibody-phage (titered at 1.1×10¹³ phage/ml) to each of eight IL-8coated wells followed by an 1 hour incubation at 25° C. Thenon-specifically bound antibody-phage were removed by 10 quick washeswith PBS-0.05% Tween 20 (PBS-Tween). For sort #1, a low stringency wash(100 ul PBS-Tween/well for 10 minutes at 25° C.) was employed to capturethe small proportion of tight binding antibody-phage bound to theimmobilized IL-8. The antibody-phage variants specifically bound to IL-8were eluted with 100 ul/well of 200 mM Glycine pH 2.0 for 5 minutes at25° C. The eluted antibody-phage variants from the 8 wells were thenpooled and neutralized with 1M Tris-HCl pH 8.0 (⅓ the elution volume).The phage were titered and propagated as described in Section (I) above.The stringency of the washes were successively increased with each roundof panning depending upon the percent recovery of phage at the end of asort. The wash conditions were as follows: sort #2 (4×15 minuteintervals; total time=60 minutes) and sort #3 (either #3a: 8×15 minuteintervals or #3b: 12×10 minute intervals; total time=120 minutes). Thetotal number of phage recovered was progressively reduced after eachsort suggesting that non- or weak-binders were being selected against.The recovery of the negative control (the antibody-phage stop variant)was constant throughout the panning (approximately 0.0001 to 0.00001percent).

Eighteen random variants from sort #3 were analyzed by DNA sequencing tolook for an amino acid consensus at position 35 of CDR-L1. The datapresented in FIG. 43A showed that Glycine occupied position 35 in 33% ofthe variants sequenced. However, after correcting for the number of NNScodon combinations/amino acid, the frequency of Glycine was reduced to16.6%. Glutamic Acid was represented with the highest frequency (22%)followed by Aspartic Acid and Glycine (16.6%). The frequencies ofrecovery of the wild-type Asparagine and substituted Alanine were only5.6%. Interestingly, the high frequency of Glycine may suggest that amuch wider range of conformations might be allowed for the loop ofCDR-L1 which may be attributed to the reduction in steric hindrance ofbond angle (φ-ψ) pairing as a result of the single hydrogen atom as theside chain. Conversely, Glutamic Acid at position 35 might restrict theflexibility of the loop by imposing less freedom of rotation imposed bythe more rigid and bulky charged polar side chain.

Soluble Fab's of the affinity matured variants (N35G, N35D, N35E andN35A) were made as described in Section (J) above for evaluating theirability to block IL-8 binding. As shown in FIGS. 43B, 43C, 43D, and 43Evariants N35A, N35D, N35E and N35G were found to inhibit ¹²⁵I-IL-8binding to human neutrophils with an approximate IC₅₀ of 0.2 nM, 0.9 nM,0.1 nM and 3.0 nM, respectively. All of the affinity matured variantsshowed an improvement in binding IL-8 ranging from 3–100 fold comparedto the humanized 6G4V11 mAb. The affinity-matured variant, 6G4V11N35E,was 2-fold more potent in blocking IL-8 binding to human neutrophilsthan the alanine-scan variant, 6G4V11N35A.

Equilibrium and kinetic measurements of variants 6G4V11N35A and6G4V11N35E were determined using KinEXA™ automated immunoassay system(Sapidyne Instruments Inc., Idaho City, Id.) as described by Blake etal., J. Biol. Chem. 271: 27677 (1996). The procedure for preparing theantigen-coated particles was modified as follows: 1 ml of activatedagarose beads (Reacti-Gel 6×; Pierce, Rockford, Ill.) were coated withantigen in 50 mM Carbonate buffer pH 9.6 containing 20 ug/ml of humanIL-8 and incubated with gentle agitation on a rocker overnight at 25° C.The IL-8 coated beads were then washed twice with 1M Tris-HCl pH 7.5 toinactivate any unreactive groups on the beads and blocked withSuperblock (Pierce, Rockford, Ill.) for 1 hour at 25° C. to reducenon-specific binding. The beads were resuspended in assay buffer (0.1%bovine serum albumin in PBS) to a final volume of 30 ml. A 550 ulaliquot of the IL-8 coated bead suspension was used each time to pack afresh 4 mm high column in the KinEXA observation cell. The amount ofunbound antibody from the antibody-antigen mixtures captured by theIL-8-coated beads in both the equilibrium and kinetic experiments wasquantified using a fluorescently labeled secondary antibody. Murine6G4.2.5 was detected with a R-PE AffiniPure F(ab′)₂ goat anti-mouse IgG,Fc fragment specific 2° antibody (Jackson Immuno Research Laboratories,West Grove, Pa.) and humanized affinity matured N35A (Fab and F(ab′)₂)and N35E Fab were detected with a R-PE AffiniPure F(ab′)₂ donkeyanti-human IgG (H+ L) 2° antibody (Jackson Immunoresearch Laboratories,West Grove, Pa.; both at a 1:1000 dilution.

Equilibrium measurements were determined by incubating a constant amountof anti-IL-8 antibody (0.005 ug/ml) with various concentrations of humanIL-8 (0, 0.009, 0.019, 0.039, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5 nM).The antibody-antigen mixture was incuabted for 2 hours at 25° C. toallow the molecules to reach equilibrium. Subsequently, each sample waspassed over a naive IL-8 coated bead pack in the KinEXA observation cellat a flow rate of 0.5 ml/minute for a total of 9 minutes/sample. Theequilibrium constant (Kd) was calculated using the software provided bySapidyne Instruments Inc.

Rates of association (ka) and dissociation (kd) were determined byincubating together a constant amount of antibody and antigen, andmeasuring the amount of uncomplexed anti-IL-8 bound to the IL-8 coatedbeads over time. The concentration of antibody used in the kineticexperiments was identical to that used in the equilibrium experimentdescribed above. Generally, the amount of human IL-8 used was theconcentration derived from the binding curves of the equilibriumexperiment that resulted in 70% inhibition of anti-IL-8 binding to theIL-8 coated beads. Measurements were made every 15 minutes to collectapproximately nine data points. The ka was calculated using the softwareprovided by Sapidyne Instruments, Inc. The off rate was determined usingthe equation: kd=Kd/ka.

FIG. 44 shows the equilibrium constants (Kd) for the affinity maturedvariants 6G4V11N35E and 6G4V11N35A Fab's were approximately 54 pM and114 pM, respectively. The improvement in affinity of 6G4V11N35E Fab forIL-8 can be attributed to a 2-fold faster rate of association (K_(on))of 4.7×10⁶ for 6G4V11N35E Fab versus 2.0×10⁶ for 6G4V11N35A F(ab′)₂.(The Kd of the 6G4V11N35A F(ab′)2 and 6G4V11N35A Fab are similar.) Thedissociation rates (K_(off)) were not significantly different. Molecularmodeling suggests that substitution of Aspargine with Glutamic Acidmight either affect the antibody's interaction with IL-8 directly orindirectly by neutralizing the charge of neighboring residues R98(CDR-H3) or K50 (CDR-L2) in the CDR's to facilitate contact with IL-8.Another effect might be the formation of a more stable loop conformationfor CDR-L1 that could have facilitated more appropriate contacts ofother CDR-L1 loop residues with IL-8. The DNA (SEQ ID NO: 65) and aminoacid (SEQ ID NO:62) sequences of p6G4V11N35E.Fab showing the Asparagineto Glutamic Acid substitution in the light chain are presented in FIG.45.

N. Characterization of Humanized Anti-IL-8 Variant 6G4V11N35E Fab

The affinity matured Fab variant, 6G4V11N35E, was tested for its abilityto inhibit IL-8 mediated neutrophil chemotaxis as described in Section(B)(2) above. The reuseable 96-well chemotaxis chamber described inSection (B)(2) was replaced with endotoxin-free disposable chemotaxischambers containing 5-micron PVP-free polycarbonate filters(ChemoTx101-5, Neuro Probe, Inc. Cabin John, Md.). As illustrated inFIG. 46, variant N35E effectively blocks IL-8 mediated neutrophilchemotaxis induced by a 2 nM stimulus of either rabbit or human IL-8. Infact, the level of inhibition at antibody concentrations between 3.7nM–33 nM was not significantly different from the buffer controlindicating variant N35E could completely inhibit this response. TheIC₅₀'s for both rabbit and human IL-8 were approximately 2.8 nM and 1.2nM, respectively. The irrelevant isotype control Fab (4D5) did notinhibit neutrophil migation indicating the results observed for theaffinity matured variant, N35E, is IL-8 specific.

O. Construction of Humanized 6G4V11N35E F(ab′)₂ Leucine Zipper

A F(ab′)₂ expression plasmid for 6G4V11N35E was constructed usingmethods similar to those described in Section (K) above. The expressionplasmid, p6G4V11N35E.F(ab′)₂, was made by digesting the plasmidp6G4V11N35A.F(ab′)₂ (described in Section (K) above) with therestriction enzymes ApaI and NdeI to isolate a 2805 bp fragment encodingthe heavy chain constant domain—GCN4 leucine zipper and ligating it to a3758 bp ApaI-NdeI fragment of the pPH6G4V11N35E phage display clone(encoding 6G4V11N35E Fab) obtained as described in Section (M) above.The integrity of the entire coding sequence was confirmed by DNAsequencing.

P. Construction of the Full Length Humanized 6G4V11N35A IgG ExpressionPlasmid

The full length IgG₁ version of the humanized anti-IL8 variant6G4V11N35A was made using a dicistronic DHFR-Intron expression vector(Lucas et al., Nucleic Acids Res., 24: 1774–1779 (1996)) which containedthe full length recombinant murine-human chimera of the 6G4.2.5 anti-IL8mAb. The expression plasmid encoding the humanized variant 6G4V11N35Awas assembled as follows. First an intermediate plasmid (pSL-3) was madeto shuttle the sequence encoding the variable heavy chain of humanizedanti-IL-8 variant 6G4V11N35A to pRK56G4chim.2Vh—which contains thevariable heavy region of the chimeric 6G4.5 anti-IL8 antibody. Thevector pRK56G4chim. Vh was digested with PvuII and ApaI to remove theheavy chain variable region of the chimeric antibody and religated withan 80 bp PvuII-XhoI synthetic oligonucleotide (encoding Leu4 to Phe29 of6G4V11N35A) (FIG. 47) and a 291 bp XhoI-ApaI fragment from p6G4V11N35A.7carrying the remainder of the variable heavy chain sequence of6G4V11N35A to create pSL-3. This intermediate plasmid was used inconjunction with 2 other plasmids, p6G4V11N35A.F(ab′)₂ andp6G425chim2.choSD, to create the mammalian expression plasmid,p6G4V11N35AchoSD.9 (identified as p6G425V11N35A.choSD in a deposit madeon Dec. 16, 1997 with the ATCC and assigned ATCC Accession No. 209552).This expression construct was assembled in a 4-part ligation using thefollowing DNA fragments: a 5,203 bp ClaI-BlpI fragment encoding theregulatory elements of the mammalian expression plasmid (p6G425chim2.choSD), a 451 bp ClaI-ApaI fragment containing the heavy chainvariable region of the humanized 6G4V11N35A antibody (pSL-3), a 1,921 bpApaI-EcoRV fragment carrying the heavy chain constant region of6G4V11N35A (p6G425chim2.choSD) and a 554 bp EcoRV-BlpI fragment encodingthe light chain variable and constant regions of 6G4V11N35A(p6G4V11N35A.F(ab′)₂). The DNA sequence (SEQ ID NO: 68) of clonep6G4V11N35A.choSD.9 was confirmed by DNA sequencing and is presented inFIG. 48.

Q. Construction of the Full Length Humanized 6G4V11N35E IgG ExpressionPlasmid

A mammalian expression vector for the humanized 6G4V11N35E was made byswapping the light chain variable region of 6G4V11N35A with 6G4V11N35Eas follows: a 7,566 bp EcoRV-BlpI fragment (void of the 554 bp fragmentencoding the light chain variable region of 6G4V11N35A) fromp6G4V11N35A.choSD.9 was ligated to a 554 bp EcoRV-BlpI fragment(encoding the light chain variable region of 6G4V11N35E) frompPH6G4V11N35E.7. The mutation at position N35 of the light chain ofp6G4V11N35E.choSD.10 was confirmed by DNA sequencing.

R. Stable CHO Cell Lines for variants N35A and N35E

For stable expression of the final humanized IgG1 variants (6G4V11N35Aand 6G4V11N35E), Chinese hamster ovary (CHO) DP-12 cells weretransfected with the above-described dicistronic vectors(p6G4V11N35A.choSD.9 and p6G4V11N35E.choSD.10, respectively) designed tocoexpress both heavy and light chains (Lucas et al., Nucleic Acid Res.24:1774–79 (1996)). Plasmids were introduced into CHO DP12 cells vialipofection and selected for growth in GHT-free medium (Chisholm, V.High efficiency gene transfer in mammalian cells. In: Glover, D M,Hames, B D. DNA Cloning 4. Mammalian systems. Oxford Univ. Press, Oxfordpp 1–41 (1996)). Approximately 20 unamplified clones were randomlychosen and reseeded into 96 well plates. Relative specific productivityof each colony was monitored using an ELISA to quantitate the fulllength human IgG accumulated in each well after 3 days and a fluorescentdye, Calcien AM, as a surrogate marker of viable cell number per well.Based on these data, several unamplified clones were chosen for furtheramplification in the presence of increasing concentrations ofmethotrexate. Individual clones surviving at 10, 50, and 100 nMmethotrexate were chosen and transferred to 96 well plates forproductivity screening. One clone for each antibody (clone#1933 aIL8.92NB 28605/12 for 6G4V11N35A; clone#1934 aIL8.42 NB 28605/14 for6G4V11N35E), which reproducibly exhibited high specific productivity,was expanded in T-flasks and used to inoculate a spinner culture. Afterseveral passages, the suspension-adapted cells were used to inoculateproduction cultures in GHT-containing, serum-free media supplementedwith various hormones and protein hydrolysates. Harvested cell culturefluid containing recombinant humanized anti-IL8 was purified usingprotein A-Sepharose CL-4B. The purity after this step was approximately99%. Subsequent purification to homogeneity was carried out using an ionexchange chromatography step. Production titer of the humanized6G4V11N35E IgG1 antibody after the first round of amplification and6G4V11N35A IgG1 after the second round of amplification were 250 mg/Land 150 mg/L, respectively.

S. Characterization of the Humanized 6G4V11N35A/E IgG Variants

The humanized full length IgG variants of 6G4.2.5 were tested for theirability to inhibit ¹²⁵I-IL-8 binding and to neutralize activation ofhuman neutrophils; the procedures are described in Sections (B)(1) and(B)(2) above. As shown in FIG. 49, the full length IgG1 forms ofvariants 6G4V11N35A and 6G4V11N35E equally inhibited ¹²⁵I-IL-8 bindingto human neutrophils with approximate IC₅₀'s of 0.3 nM and 0.5 nM,respectively. This represents a 15–25 fold improvement in blockingbinding of IL-8 compared to the full length murine mAb (IC₅₀=7.5 nM).Similarly, the two anti-IL-8 variants showed equivalent neutralizingcapabilities with respect to inhibiting IL-8 mediated human neutrophilchemotaxis (FIGS. 50A–50B). The IC₅₀'s of 6G4V11N35A IgG1 and 6G4V11N35EIgG1 for human IL-8 were 4.0 nM and 6.0 nM, respectively, and for rabbitIL-8 were 4.0 nM and 2.0 nM, respectively. The irrelevant isotypecontrol Fab (4D5) did not inhibit neutrophil migration.

The affinity for IL-8 of these variants relative to the murine 6G4.2.5mAb was determined using KinExA as described in Section (M). FIG. 51shows the equilibrium constant (Kd) for the full length affinity maturedvariants 6G4V11N35E IgG1 and 6G4V11N35A IgG1 were approximately 49 pMand 88 pM, respectively. The Kd for 6G4V11N35A IgG1 was determineddirectly from the kinetic experiment. As reported with their respectiveFabs, this improvement in affinity might be attributed to an approximate2-fold increase in the on-rate of 6G4V11N35E IgG1 (ka=3.0×10⁶) comparedto that of 6G4V11N35A IgG1 (ka=8.7×10⁵). In addition, these results wereconfirmed by a competition radio-immune assay using iodinated humanIL-8. 50 pM of 6G4V11N35A IgG1 or 6G4V11N35E IgG1 was incubated for 2hours at 25° C. with 30–50 pM of ¹²⁵I-IL-8 and varying concentrations (0to 100 nM) of unlabeled IL-8. The antibody-antigen mixture was thenincubated for 1 hour at 4° C. with 10 ul of a 70% slurry of Protein-Abeads (pre-blocked with 0.1% BSA). The beads were briefly spun in amicrocentrifuge and the supernatant discarded to remove the unbound¹²⁵I-IL-8. The amount of ¹²⁵I-IL-8 specifically bound to the anti-IL-8antibodies was determined by counting the protein-A pellets in a gammacounter. The approximate Kd values were similar to those determined byKinEXA. The average Kd for 6G4V11N35A IgG1 and 6G4V11N35E IgG1 were 54pM (18–90 pM) and 19 pM (5–34 pM), respectively (FIG. 52).

T. Construction of Humanized 6G4V11N35A/E Fab's for Modification byPolyethylene Glycol

A Fab′ expression vector for 6G4V11N35A was constructed by digestingp6G4V11N35A.F(ab′)₂ with the restriction enzymes ApaI and NdeI to removethe 2805 bp fragment encoding the human IgG₁ constant domain fused withthe yeast GCN4 leucine zipper and replacing it with the 2683 bpApaI-NdeI fragment from the plasmid pCDNA.18 described in Eigenbrot etal., Proteins: Struct. Funct. Genet., 18: 49–62 (1994). The pCDNA.18ApaI-NdeI fragment carries the coding sequence for the human constantIgG1 heavy domain, including the free cysteine in the hinge region thatwas used to attach the PEG molecule. The 3758 bp ApaI-NdeI fragment(encodes the light chain and heavy variable domain of 6G4V11N35A)isolated from p6G4V11N35A.F(ab′)₂ was ligated to the 2683 bp ApaI-NdeIfragment of pCDNA.18 to create p6G4V11N35A.PEG-1. The integrity of theentire coding sequence was confirmed by DNA sequencing. The nucleotideand translated amino acid sequences of heavy chain constant domain withthe cysteine in the hinge are presented in FIG. 53.

A Fab′ expression plasmid for 6G4V11N35E was made similarly by digestingpPH6G4V11N35E (from Section (O) above) with the restriction enzymes ApaIand NdeI to isolate the 3758 bp ApaI-NdeI DNA fragment carrying theintact light chain and heavy variable domain of 6G4V11N35E and ligatingit to the 2683 bp ApaI-NdeI DNA fragment from p6G4V11N35A.PEG-1 tocreate p6G4V11N35E.PEG-3. The integrity of the entire coding sequencewas confirmed by DNA sequencing.

Anti-IL-8 6G4V11N35A Fab′ variant was modified with 20 kD linearmethoxy-PEG-maleimide, 30 kD linear methoxy-PEG-maleimide, 40 kD linearmethoxy-PEG-maleimide, or 40 kD branched methoxy-PEG-maleimide asdescribed below. All PEG's used were obtained commercially fromShearwater Polymers, Inc.

a. Materials and Methods

Fab′-SH Purification

A Fab′-SH antibody fragment of the affinity matured antibody 6G4V11N35Awas expressed in E. coli grown to high cell density in the fermentor asdescribed by Carter et al., Bio/Technology 10, 163–167 (1992).Preparation of Fab′-SH fragments was accomplished by protecting theFab′-SH fragments with 4′,4′-dithiodipyridine (PDS), partially purifyingthe protected Fab′-PDS fragments, deprotect the Fab′-PDS withdithiothreitol (DTT) and finally isolate the free Fab′-SH by using gelpermeation chromatography.

Protection of Fab′-SH with PDS

Fermentation paste samples were dissolved in 3 volumes of 20 mM MES, 5mM EDTA, pH 6.0 containing 10.7 mg of 4′,4′-dithiodipyridine per gramfermentation paste, resulting in a suspension with a pH close to 6.0 Thesuspension was passed through a homogenizer followed by addition of 5%PEI (w/v), pH 6 to the homogenate to a final concentration of 0.25%. Themixture was then centrifuged to remove solids and the clear supernatantwas conditioned to a conductivity of less than 3 mS by the addition ofcold water.

Partial Purification of the Fab′-SH Molecule Using Ion ExchangeChromatography

The conditioned supernatant was loaded onto an ABX (Baker) columnequilibrated in 20 mM MES, pH 6.0. The column was washed with theequilibration buffer followed by elution of the Fab′-SH with a 15 columnvolume linear gradient from 20 mM MES, pH 6.0 to 20 mM MES, 350 mMsodium chloride. The column was monitored by absorbance at 280 nm, andthe eluate was collected in fractions.

Deprotection of the Fab′-SH Antibody Fragments with DTT

The pH of the ABX pool was adjusted to 4.0 by the addition of diluteHCl. The pH adjusted solution was then deprotected by adding DTT to afinal concentration of 0.2 mM. The solution was incubated for about 30minutes and then applied to a gel filtration Sephadex G25 column,equilibrated with 15 mM sodium phosphate, 25 mM MES, pH 4.0. Afterelution, the pH of the pool was raised to pH 5.5 and immediately flashfrozen at −70° C. for storage or derivatized with PEG-MAL as describedbelow.

Alternative Fab′-SH Purification

Alternatively Fab′-SH fragments can be purified using the followingprocedure. 100 g fermentation paste is thawed in the presence of 200 ml50 mM acetic acid, pH 2.8, 2 mM EDTA, 1 mM PMSF. After mixing vigorouslyfor 30 min at room temperature, the extract is incubated with 100 mg henegg white lysozyme. DEAE fast flow resin (approximately 100 mL) isequilibrated with 10 mM MES, pH 5.5, 1 mM EDTA on a sintered glassfunnel. The osmotic shock extract containing the Fab′-SH fragment isthen filtered through the resin.

A protein G Sepharose column is equilibrated with 10 mM MES, pH 5.5, 1mM EDTA and then loaded with the DEAE flow-through sample. The column iswashed followed by three 4 column volume washes with 10 mM MES, pH 5.5,1 mM EDTA. The Fab′-SH antibody fragment containing a free thiol iseluted from the column with 100 mM acetic acid, pH 2.8, 1 mM EDTA. Afterelution, the pH of the pool is raised to pH 5.5 and immediately flashfrozen at −70° C. for storage or derivatized with PEG-MAL as describedbelow.

Preparation of Fab′-S-PEG

The free thiol content of the Fab′-SH preparation obtained as describedabove was determined by reaction with 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB) analysis according to the method of Creighton in ProteinStructure: A Practical Approach, Creighton, T. E., ed, IRL Press(Oxford, UK: 1990), pp. 155–167. The concentration of free thiol wascalculated from the increase on absorbance at 412 nm, using e₄₁₂=14,150cm⁻¹ M⁻¹ for the thionitrobenzoate anion and a M_(r)=48,690 and e₂₈₀=1.5for the Fab′-SH antibody. To the Fab′-SH protein G Sepharose pool, orthe deprotected Fab′-SH gel permeation pool, 5 molar equivalents ofPEG-MAL were added and the pH was immediately adjusted to pH 6.5 with10% NaOH.

The Fab′-S-PEG was purified using a 2.5×20 cm cation exchange column(Poros 50-HS). The column was equilibrated with a buffer containing 20mM MES, pH 5.5. The coupling reaction containing the PEGylated antibodyfragment was diluted with deionized water to a conductivity ofapproximately 2.0 mS. The conditioned coupling reaction was then loadedonto the equilibrated Poros 50 HS column. Unreacted PEG-MAL was washedfrom the column with 2 column volumes of 20 mM MES, pH 5.5. TheFab′-S-PEG was eluted from the column using a linear gradient from 0 to400 mM NaCl, in 20 mM MES pH 5.5, over 15 column volumes.

Alternatively a Bakerbond ABX column can be used to purify theFab′-S-PEG molecule. The column is equilibrated with 20 mM MES, pH 6.0(Buffer A). The coupling reaction is diluted with deionized water untilthe conductivity equaled that of the Buffer A (approximately 2.0 mS) andloaded onto the column. Unreacted PEG-MAL is washed from the column with2 column volumes of 20 mM MES, pH 6.0. The Fab′-S-PEG is eluted from thecolumn using a linear gradient from 0 to 100 mM (NH₄)₂SO₄, in 20 mM MESpH 6.0, over 15 column volumes.

Size Exclusion Chromatography

The hydrodynamic or effective size of each molecule was determined usinga Pharmacia Superose-6 HR 10/30 column (10×300 mm). The mobile phase was200 mM NaCl, 50 mM sodium phosphate pH 6.0. Flow rate was at 0.5 ml/minand the column was kept at ambient temperature. Absorbance at 280 nm wasmonitored where PEG contributed little signal. Biorad MW standardscontaining cyanocobalamin, myoglobin, ovalbumin, IgG, Thyroglobulinmonomer and dimer were used to generate a standard curve from which theeffective size of the pegylated species was estimated.

b. Results

Size Exclusion Chromatography

The effective size of each modified species was characterized using sizeexclusion chromatography. The results are shown in FIG. 60 below. Thetheoretical molecular weight of the anti-IL8 Fab fragments modified withPEG 5 kD, 10 kD, 20 kD, 30 kD, 40 kD (linear), 40 kD (branched) or100,000 kD is shown along with the apparent molecular weight of thePEGylated fragments obtained by HPLC size exclusion chromatography. Whencompared to the theoretical molecular weight of the Fab′-S-PEGfragments, the apparent molecular weight (calculated by size exclusionHPLC) increases dramatically by increasing the size of the PEG attachedto the fragments. Attachment of a small molecular weight PEG, forexample PEG 10,000 D only increases the theoretical molecular weight ofthe PEGylated antibody fragment (59,700 D) by 3 fold to an apparentmolecular weight of 180,000 D. In contrast attachment of a largermolecular weight PEG for example 100,000 D PEG to the antibody fragmentincreases the theoretical molecular weight of the PEGylated antibodyfragment (158,700 D) by 12 fold to an apparent molecular weight of2,000,000 D.

SDS-Page

In FIG. 61, the upper panel shows the size of the anti-IL-8 Fabfragments modified with PEG of molecular weight 5 kD (linear), 10 kD(linear), 20 kD (linear), 30 kD (linear), 40 kD (linear), 40 kD(branched) or 100 kD (linear) under reduced conditions. The unmodifiedFab is shown in lane 2 from right to left. Both the heavy and lightchains of the Fab had a molecular weight of approximately 30 kD asdetermined by PAGE. Each PEGylated fragment sample produced two bands:(1) a first band (attributed to the light chain) exhibiting a molecularweight of 30 kD; and (2) a second band (attributed to the heavy chain towhich the PEG is attached specifically at the hinge SH) exhibitingincreasing molecular weights of 40, 45, 70, 110, 125, 150 and 300 kD.This result suggested that PEGylation was specifically restricted to theheavy chain of the Fab's whereas the light chain remained unmodified.

The lower panel is non-reduced PAGE showing the size of the anti-IL-8Fab fragments modified with PEG of molecular weight 5 kD (linear), 20 kD(linear), 30 kD (linear), 40 kD (linear), 40 kD (branched), or 100 kD(linear). The PEGylated fragments exhibited molecular weights ofapproximately 70 kD, 115 kD, 120 kD, 140 kD, 200 kD and 300 kD.

The SDS PAGE gels confirm that all Fab′-S-PEG molecules were purified tohomogeneity and that the molecules differed only with respect to thesize of the PEG molecule attached to them.

U. Amine Specific Pegylation of Anti-IL-8 F(ab′)₂ Fragments

Pegylated F(ab′)₂ species were generated by using large MW or branchedPEGs in order to achieve a large effective size with minimal proteinmodification which might affect activity. Modification involvedN-hydroxysuccinamide chemistry which reacts with primary amines (lysinesand the N-terminus). To decrease the probability of modifying theN-terminus, which is in close proximity to the CDR region, a reaction pHof 8, rather than the commonly used pH of 7, was employed. At pH 8.0,the amount of the reactive species (charged NH₃ ⁺) would be considerablymore for the ε-NH2 group of lysines (pK_(a)=10.3) than for the α-NH2group (pK_(a) of approximately 7) of the amino-terminus. For the linearPEGs, a methoxy-succinimidyl derivative of an NHS-PEG was used becauseof the significantly longer half-life in solution (17 minutes at 25° C.at pH 8.0) compared to the NHS esters of PEGs (which have 5–7 minutehalf life under the above conditions). By using a PEG that is less proneto hydrolysis, a greater extent of modification is achieved with lessPEG. Branched PEGs were used to induce a large increase in effectivesize of the antibody fragments.

a. Materials

All PEG reagents were purchased from Shearwater Polymers and stored at−70° C. in a desiccator: branched N-hydroxysuccinamide-PEG (PEG2-NHS-40KDa) has a 20 kDa PEG on each of the two branches,methoxy-succinimidyl-propionic acid-PEG (M-SPA-20000) is a linear PEGmolecule with 20 kDa PEG. Protein was recombinantly produced in E. coliand purified as a (Fab)′₂ as described in Sections (K) and (O) above.

b. Methods

IEX method: A J. T. Baker Wide-Pore Carboxy-sulfone (CSX), 5 micron,7.75×100 mm HPLC column was used for fractionation of the differentpegylated products, taking advantage of the difference in charge as thelysines are modified. The column was heated at 40° C. A gradient asshown in Table 7 below was used where Buffer A was 25 mM sodiumBorate/25 mM sodium phosphate pH 6.0, and Buffer B was 1 M ammoniumsulfate, and Buffer C was 50 mM sodium acetate pH 5.0.

TABLE 7 Time (min) % B % C flow mL/min 0 10 10 1.5 20 18 7.5 1.5 25 257.5 1.5 27 70 3.0 2.5 29 70 3.0 2.5 30 10 10 2.5 33 10 10 2.5

SEC-HPLC: The hydrodynamic or effective size of each molecule wasdetermined using a Pharmacia Superose-6 HR 10/30 column (10×300 mm). Themobile phase was 200 mM NaCl, 50 mM sodium phosphate pH 6.0. Flow ratewas at 0.5 ml/min and the column was kept at ambient temperature.Absorbance at 280 nm was monitored where PEG contributed little signal.Biorad MW standards containing cyanocobalamin, myoglobin, ovalbumin,IgG, Thyroglobulin monomer and dimer were used to generate a standardcurve from which the effective size of the pegylated species wasestimated.

SEC-HPLC-Light Scattering: For determination of the exact molecularweight, this column was connected to an on-line light scatteringdetector (Wyatt Minidawn) equipped with three detection angles of 50°,90°, and 135° C. A refractive index detector (Wyatt) was also placedon-line to determine concentration. All buffers were filtered withMillipore 0.1μ filters; in addition al 0.02μ Whatman Anodisc 47 wasplaced on-line prior to the column.

The intensity of scattered light is directly proportional to themolecular weight (M) of the scattering species, independent of shape,according to:M=R ₀ /K.cwhere R₀ is the Rayleigh ratio, K is an optical constant relating to therefractive index of the solvent, the wavelength of the incident light,and dn/dc, the differential refractive index between the solvent and thesolute with respect to the change in solute concentration, c. The systemwas calibrated with toluene (R₀ of 1.406×10⁻⁵ at 632.8 nm); a dn/dc of0.18, and an extinction coefficient of 1.2 was used. The system had amass accuracy of ˜5%.

SDS-PAGE: 4–12% Tris-Glycine Novex minigels were used along with theNovex supplied Tris-Glycine running buffers. 10–20 ug of protein wasapplied in each well and the gels were run in a cold box at 150 mV/gelfor 45 minutes. Gels were then stained with colloidal Coomassie Blue(Novex) and then washed with water for a few hours and then preservedand dried in drying buffer (Novex)

Preparation of a linear(1)20 KDa-(N)-(Fab′)2: A 4 mg/ml solution ofanti-IL8 formulated initially in a pH 5.5 buffer was dialyzed overnightagainst a pH 8.0 sodium phosphate buffer. 5 mL protein was mixed at amolar ratio of 3:1. The reaction was carried out in a 15 mLpolypropylene Falcon tube and the PEG was added while vortexing thesample at low speed for 5 seconds. It was then placed on a nutator for30 minutes. The extent of modification was evaluated by SDS-PAGE. Thewhole 5 ml reaction mixture was injected on the IEX for removal of anyunreacted PEG and purification of singly or doubly pegylated species.The above reaction generated a mixture of 50% singly-labeled anti-IL8.The other 50% unreacted anti-IL8 was recycled through thepegylation/purification steps. The pooled pegylated product was dialyzedagainst a pH 5.5 buffer for in vitro assays and animal PK studies.Endotoxin levels were measured before administration to animals or forthe cell based assays. Levels were below 0.5 eu/ml. The fractions werealso run on SDS-PAGE to confirm homogeneity. Concentration of the finalproduct was assessed by absorbance at 280 nm using an extinctioncoefficient of 1.34, as well as by amino acid analysis.

Preparation of a branched(1)40 KDa-(N)-(Fab′)2: A 4 mg/mL solution ofanti-IL8 (Fab′)₂ formulated in a pH 5.5 buffer was dialyzed overnightagainst a pH 8.0 phosphate buffer. Solid PEG powder was added to 5 mLprotein in two aliquots to give a final PEG:protein molar ratio of 6:1.Each solid PEG aliquot was added to the protein in a 15 mL polypropyleneFalcon tube while vortexing at low speed for 5 sec, and then placing thesample on a nutator for 15 minutes. The extent of modification wasevaluated by SDS-PAGE using a 4–12% Tris-Glycine (Novex) gel and stainedwith colloidal Coomasie blue (Novex). The 5 mL PEG-protein mixture wasinjected on the ion exchange column for removal of any unreacted PEG.The above reaction generated a mixture of unreacted (37%),singly-labelled (45%), doubly and triply-labeled (18%) species. Thesewere the optimal conditions for obtaining the greatest recovery of theprotein with only 1 PEG per antibody rather than the higher molecularweight adducts. The unmodified anti-IL8 was recycled. The pegylatedproducts were separated and fractionated in falcon tubes and thendialyzed against a pH 5.5 buffer for assays and animal PK studies.Endotoxin levels were below 0.5 eu/ml. The fractions were also run onSDS-PAGE to confirm homogeneity. The concentration of the final productwas assessed by absorbance at 280 nm using an extinction coefficient of1.34, as well as by amino acid analysis.

Preparation of branched(2)-40 KDa-(N)(Fab′)2: This molecule was mostefficiently made by adding three times in 15 minute intervals a 3:1molar ratio of PEG to the already modified branched(1)-40KDa-(N)-(Fab′)2. The molecule was purified on IEX as 50% branched(2)-40KDa-(N)-(Fab′)2. The unmodified molecule was recycled until ˜20 mgprotein was isolated for animal PK studies. The product wascharacterized by SEC-light scattering and SDS-PAGE.

c. Results

PEGs increased the hydrodynamic or effective size of the productsignificantly as determined by gel filtration (SEC-HPLC). FIG. 62 showsthe SEC profile of the pegylated F(ab′)₂ species with UV detection at280 nm. The hydrodynamic size of each molecule was estimated byreference to the standard MW calibrators. As summarized in FIG. 62, theincrease in the effective size of (Fab′)₂ was about 7-fold by adding onelinear 20 kDa PEG molecule and about 11-fold by adding one branched(“Br(1)”) 40 kDa PEG molecule, and somewhat more with addition of twobranched (“Br(2)”) PEG molecules.

Light scattering detection gave the exact molecular weight of theproducts and confirmed the extent of modification (FIG. 63). Thehomogeneity of the purified material was shown by SDS-PAGE (FIG. 64).Underivatized F(ab′)₂ migrated as a 120 kDa species, the linear(1)20KD-(N)-F(ab′)₂ migrated as a band at 220 kDa, the Br(1)-40 KD(N)-F(ab′)₂migrated as one major band at 400 kDa, and the Br(2)-40 KD-(N)-F(ab′)₂migrated as a major band at around 500 kDa. The proteins appearedsomewhat larger than their absolute MW due to the steric effect of PEG.

V. In Vitro Activity Characterization of PEG Modified Fab′ Fragments of6G4V11N35A (Maleimide Chemical Coupling Method)

Anti-IL-8 6G4V11N35A Fab′ variants modified with 5–40 kD linear PEGmolecules and a 40 kD branched PEG molecule were tested for theirability to inhibit both IL-8 binding and activation of humanneutrophils; the procedures were described in Sections (B)(1), (B)(2)and (B)(3) above. The binding curves and IC₅₀'s for PEG-maleimidemodified 6G4V11N35A Fab′ molecules are presented in FIGS. 54A–54C. TheIC₅₀ of the 5 kD pegylated Fab′ (350 pM) and the average IC₅₀ of the Fabcontrol (366 pM) were not significantly different, suggesting that theaddition of a 5 kD MW PEG did not affect the binding of IL-8 to themodified Fab′ (FIG. 54A). However, a decrease in the binding of IL-8 tothe 10 kD and 20 kD pegylated Fab′ molecules was observed as depicted bythe progressively higher IC₅₀'s (537 pM and 732 pM, respectively)compared to the average IC₅₀ of the native Fab. These values representonly a minimal loss of binding activity (between 1.5- and 2.0-fold). Aless pronounced difference in IL-8 binding was observed for the 30 kDand 40 kD linear PEG antibodies (FIG. 54B). The IC₅₀'s were 624 pM and1.1 nM, respectively, compared to the 802 pM value of the Fab control.The 40 kD branched PEG Fab′ showed the largest decrease in IL-8 binding(2.5 fold) relative to the native Fab (FIG. 54C). Nevertheless, thereduction in binding of IL-8 by these pegylated Fab's is minimal.

The ability of the pegylated antibodies to block IL-8 mediatedactivation of human neutrophils was demonstrated using the PMNchemotaxis (according to the method described in Section B(2) above) andβ-glucuronidase release (according to the method described in Lowman etal., J. Biol. Chem., 271: 14344 (1996)) assays. The IC₅₀'s for blockingIL-8 mediated chemotaxis are shown in FIGS. 55A–55C. The 5–20 kD linearpegylated Fab′ antibodies were able to block IL-8 mediated chemotaxiswithin 2–3 fold of the unpegylated Fab control (FIG. 55A). Thisdifference is not significant because the inherent variation can be upto 2 fold for this type of assay. However, a significant difference wasdetected for the 30 kD and 40 kD linear pegylated Fab′ antibodies asillustrated by the higher IC₅₀'s of the 30 kD linear PEG-Fab′ (2.5 nM)and 40 kD linear PEG-Fab′ (3.7 nM) compared to the Fab control (0.8 nM)(FIG. 55B). The ability of the 40 kD branched PEG Fab′ molecule to blockIL-8 mediated chemotaxis was similar to that of the 40 kD linear PEGFab′ (FIG. 55C). At most, the ability of the pegylated Fab′ antibodiesto block IL-8 mediated chemotaxis was only reduced 2–3 fold.Furthermore, release of β-glucuronidase from the granules of neutrophilswas used as another criteria for assessing IL-8 mediated activation ofhuman PMNs. FIG. 56A (depicting results obtained with 5 kD, 10 kD and 20kD linear PEGs), FIG. 56B (depicting results obtained with 30 kD and 40kD linear PEGs), and FIG. 56C (depicting results obtained with 40 kDbranched PEG) show that all the pegylated Fab′ antibodies were able toinhibit IL-8 mediated release of β-glucuronidase as well as or betterthan the unpegylated Fab control. The data collectively shows that thepegylated Fab′ variants are biological active and are capable ofinhibiting high amounts of exogenous IL-8 in in-vitro assays using humanneutrophils.

W. In Vitro Activity Characterization of Peg Modified F(ab′)₂ Fragmentsof 6G4V11N35A (Succinimidyl Chemical Coupling Method)

The anti-IL-8 variant 6G4V11N35A F(ab′)₂ modified with (a) a single 20kD linear PEG molecule per F(ab′)₂, (b) a single 40 kD branched PEGmolecule per F(ab′)₂, (c) with three, four, or five 20 kD linear PEGmolecules per F(ab′)₂ (a mixture of: (1) species having three 20 kDlinear PEG molecules per F(ab′)₂; (2) species having four 20 kD linearPEG molecules per F(ab′)₂; and (3) species having five 20 kD linear PEGmolecules per F(ab′)₂; denoted as “20 kD linear PEG (3,4,5) F(ab′)₂”),or (d) with two 40 kD branched PEG molecules per F(ab′)₂ (denoted as “40kD branch PEG (2) F(ab′)₂”), were tested for their ability to inhibit¹²⁵I-IL-8 binding and to neutralize activation of human neutrophils. Theprocedures used are described in Sections (B)(1), (B)(2) and (B)(3)above. The binding curves for pegylated F(ab′)₂ variants are shown inFIGS. 57A–57B. No significant differences were observed amongst theF(ab′)₂ control, the single 20 kD linear PEG-modified F(ab′)₂, and thesingle 40 kD branched PEG-modified F(ab′)₂ (FIG. 57A). However, theF(ab′)₂ variants containing multiple PEG molecules showed a slightreduction (less than 2-fold) in their ability to bind IL-8. The IC₅₀'sof the 20 kD linear PEG (3,4,5) F(ab′)₂ and 40 kD branch PEG (2) F(ab′)₂variants were 437 pM and 510 pM, respectively, compared to 349 pM of theF(ab′)₂ control (FIG. 57B).

The ability of these pegylated F(ab′)₂ variants to block IL-8 mediatedneutrophil chemotaxis is presented in FIGS. 58A–58B. Consistent with thePMN binding data, the single linear and branched PEG F(ab′)₂ variantswere able to block IL-8 mediated chemotaxis similar to the unpegylatedF(ab′)₂ control (FIG. 58A). The ability of the 40 kD branch PEG (2)F(ab′)₂ variant to inhibit PMN chemotaxis was identical to the controlF(ab′)₂ while the 20 kD linear PEG (3,4,5) F(ab′)₂ mixture was able toinhibit within 3-fold of the control antibody (FIG. 58B).

Shown in FIGS. 59A and 59B are the results of the β-glucuronidaserelease assay which is a measure of degranulation by IL-8 stimulatedhuman neutrophils. The single 20 kD linear PEG-modified F(ab′)₂ and thesingle 40 kD branched PEG-modified F(ab′)₂ variants were able to inhibitrelease of α-glucuronidase as well as the F(ab′)₂ control (FIG. 59A).The 40 kD branch PEG (2) F(ab′)₂ inhibited this response within 2-foldof the F(ab′)₂ control (FIG. 59B). The 20 kD linear PEG (3,4,5) moleculewas not tested. Overall, the F(ab′)₂ pegylated anti-IL-8 antibodies werebiologically active and effectively prevented IL-8 binding to humanneutrophils and the signaling events leading to cellular activation.

X. Pharmacokinetic and Safety Study of Eight Constructs of PegylatedAnti-IL-8 (Humanized) F(ab′)2 and Fab′ fragments in Normal RabbitsFollowing Intravenous Administration

The objective of this study was to evaluate the effect of pegylation onthe pharmacokinetics and safety of six pegylated humanized anti-IL-8constructs (pegylated 6G4V11N35A.Fab′ and pegylated 6G4V11N35A.F(ab′)₂obtained as described in Sections (T) and (U) above) relative to thenon-pegylated fragments in normal rabbits. Eight groups of two/threemale rabbits received equivalent protein amounts of pegylated6G4V11N35A.Fab′ or pegylated 6G4V11N35A.F(ab′)₂ constructs (2 mg/kg) viaa single intravenous (IV) bolus dose of one anti-IL8 construct. Serumsamples were collected according to the schedule shown in Table 8 belowand analyzed for anti-IL8 protein concentrations and antibody formationagainst anti-IL8 constructs by ELISA.

TABLE 8 Group Dose level/ No. Route Material Blood Collection 1 2 mg/kgFab′ control 0, 5, 30 min; 1, 2, 3, 4, 6, 8, 10, (protein conc.) 14, 20,24, 360 hr 2 IV bolus linear(1)20K(s)Fab′ 0, 5, 30 min; 1, 2, 4, 6, 8,10, 12, 3 linear(1)40K(s)Fab′ 24, 28, 32, 48, 72, 96, 168, 216, 4branched(1)40K(N)F(ab′)₂ 264, 336, 360 hr 5 F(ab′)₂ control 0, 5, 30min; 1, 2, 4, 6, 8, 10, 12, 24, 28, 32, 48, 52, 56, 336 hr 6branched(2)40K(s)Fab′ 0, 5, 30 min; 1, 2, 4, 6, 8, 10, 12, 24, 28, 32,48, 72, 96, 168, 216, 264, 336 hr; Day 17, 21, 25 7branched(2)40K(N)F(ab′)₂ 0, 5, 30 min; 1, 2, 4, 6, 8, 10, 12, 24, 28,32, 48, 72, 144, 192, 240 hr; Day 13, 16, 20, 23 8 linear(1)30K(s)Fab′0, 5, 30 min; 1, 2, 4, 6, 8, 10, 12, 24, 28, 32, 48, 72, 96, 168, 216,264, 336 hr; Day 17, 21, 25

a. Methods

Three male New Zealand White (NZW) rabbits per group (with exception toGroup 7, n=2) received an equivalent amount of 6G4V11N35A protein (Fab′or F(ab′)₂) construct at 2 mg/kg via an IV bolus dose in a marginal earvein. Amino acid composition analysis and absorbance at 280 nm usingextinction coefficients of 1.26 for 6G4V11N35A Fab′ constructs and 1.34for 6G4V11N35A F(ab′)₂ constructs were performed to determine theprotein concentration. Whole blood samples were collected via an earartery cannulation (ear opposing dosing ear) at the above time points.Samples were harvested for serum and assayed for free 6G4V11N35A Fab′ orF(ab′)₂ constructs using an IL-8 Binding ELISA. Assays were conductedthroughout the study as samples became available. All animals weresacrificed following the last blood draw, and necropsies were performedon all animals in Groups 1, 4–8. Due to the development of antibodiesagainst the 6G4V11N35A constructs, non-compartmental pharmacokineticanalysis was conducted on concentration versus time data only up to 168hours.

b. Results

In four animals (Animals B, P, Q, V), interference to rabbit serum inthe ELISA assay was detected (i.e. measurable concentrations of anti-IL8antibodies at pre-dose). However, because these values were atinsignificant levels and did not effect the pharmacokinetic analysis,the data were not corrected for this interference.

One animal (Animal G; Group 3) was exsanguinated before the terminationof the study and was excluded from the pharmacokinetic analysis. At 4hours, the animal showed signs of a stroke that was not believed to bedrug related, as this can occur in rabbits following blood draws via earartery cannulation.

The mean concentration-time profiles of the eight anti-IL8 constructs innormal rabbits are depicted in FIG. 65, and the pharmacokineticparameters for the eight constructs are summarized in Table 9 below.Significant antibodies to the anti-IL-8 constructs were present at Day13/14 in all dose groups except Group 1 (Fab′ control).

TABLE 9 Pharmacokinetic parameters. Molecule Fab′ F(ab′)₂ Group No. 1 28 3 6 5 4 7 PEG structure — linear linear linear branched — branchedbranched Number of PEGs — 1 1 1 1 — 1 2 PEG MW — 20K 30K 40K 40K — 40K40K Dose (mg/kg) 2 2 2 2 2 2 2 2 V_(c) (mL/kg)^(a) 58 ± 3 36 ± 3 35 ± 134 44 ± 1 45 ± 5 36 ± 1 32 V_(ss) (mL/kg)^(b) 68 ± 8 80 ± 8 110 ± 15 79 88 ± 21 59 ± 4 50 ± 3 52 Cmax (μg/mL)^(c) 35 ± 1 58 ± 3 57 ± 1 60 45 ±1 45 ± 6 56 ± 2 62 Tmax (min)^(d) 5 5 5 5 5 5 5 5 t_(1/2) term (hr)^(e) 3.0 ± 0.9 44 ± 2 43 ± 7 50 105 ± 11  8.5 ± 2.1 45 ± 3 48 AUC_(0–8) (hr· μg/mL)^(f) 18 ± 3  80 ± 74  910 ± 140 1600  3400 ± 1300 140 ± 3  2200± 77  2500 CL (mL/hr/kg)^(g) 110 ± 17  2.5 ± 0.2  2.2 ± 0.4 1.3  0.63 ±0.20 14 ± 0  0.92 ± 0.03 0.83 MRT (hr)^(h)  0.61 ± 0.15 32 ± 2 45 ± 9 63140 ± 18  4.2 ± 0.3 55 ± 3 64 No. of Animals 3 3 3 2 3 3 3 2 ^(a)Initialvolume of distribution. ^(b)Volume of distribution at steady state.^(c)Observed maximum concentration. ^(d)Observed time to Cmax.^(e)t_(1/2) term = half-life associated with the terminal phase of theconcentration vs. time profile. ^(f)Area under the concentration versustime curve (extrapolated to infinity). ^(g)CL = serum clearance. ^(h)MRT= Mean residence time.

The initial volume of distribution approximated the plasma volume forboth the Fab′ and F(ab′)₂. Pegylation decreased serum CL of anti-IL8fragments and extended both the terminal half-life and MRT as shown inTable 10 below.

TABLE 10 Fold decrease/increase in clearance, terminal half-life & MRTof pegylated anti-IL8 fragments. anti-IL8 fragment Fab′ F(ab′)₂ GroupNo. 1 2 8 3 6 5 4 7 PEG structure — linear linear linear bran. — bran.bran. No. of PEGs — 1 1 1 1 — 1 2 PEG MW — 20K 30K 40K 40K — 40K 40K CL:mean (mL/hr/kg) 110 2.5 2.2 1.3 0.63 14 0.92 0.83 fold decrease 1 46 5190 180 1 15 17 t½ term: mean (hr) 3.0 44 43 50 110 8.5 45 48 foldincrease 1 14 14 17 35 1 5.3 5.7 MRT: mean (hr) 0.61 32 45 63 140 4.2 5564 fold increase 1 53 73 100 240 1 13 15

For the pegylated anti-IL8 Fab′ fragments, CL decreased by 46 to180-fold. Terminal half-life and MRT increased 14 to 35-fold and 53 to240-fold, respectively. For pegylated anti-IL8 F(ab′)₂ molecules, CLdecreased 15 to 17-fold with pegylation, and terminal half-life and MRTincreased by greater than 5-fold and 13-fold, respectively. The changesin these parameters increased for both pegylated Fab′ and F(ab′)₂molecules with increasing PEG molecular weight and approached the valuesof the full-length anti-IL8 (terminal half-life of 74 hours, MRT of 99hours and CL of 0.47 mL/hr/kg). In comparing the branched(1)40K Fab′(Group 6) and branched(1)40K F(ab′)₂ (Group 4), unexpectedpharmacokinetics were observed. The pegylated Fab′ molecule appeared toremain in the serum longer than the pegylated F(ab′)₂ (see FIG. 66). Themean CL of branched(1)40K Fab′ was 0.63 mL/hr/kg, but a higher CL wasobserved for branched(1)40 kD F(ab′)₂ (CL 0.92 mL/hr/kg). The terminalhalf-life, likewise, was longer for the Fab′ than the F(ab′)₂ pegylatedmolecule (110 vs 45 hours).

The pharmacokinetic data demonstrated that pegylation decreased CL andincreased terminal t1/2 and MRT of anti-IL8 fragments (Fab′ and F(ab′)₂)to approach that of the full-length anti-IL8. Clearance was decreasedwith pegylation 46 to 180-fold for the Fab′ and approximately 16-foldfor the F(ab′)₂. The terminal half-life of the Fab′ anti-IL8 fragmentwas increased by 14 to 35-fold and approximately 5-fold for the F(ab′)₂anti-IL8. MRT, likewise, were extended by 53 to 240-fold for the Fab′and approximately 14-fold for the F(ab′)₂. The branched(1) 40 kD Fab′had a longer terminal half-life and lower clearance compared to thebranched(1) 40 kD F(ab′)₂.

Y. In Vivo Efficacy Testing of Anti-IL-8 Antibody Reagents in RabbitModel of Ischemia/Reperfusion and Acid Aspiration-Induced AcuteRespiratory Distress Syndrome (ARDS)

Full length murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5, 40 kDbranched PEG-6G4V11N35A Fab′, and control antibody (anti-HIV gp120monoclonal antibody 9E3.1F10) were tested in a rabbit ARDS model. Theanimals were weighed and anaesthetized by intramuscular injection ofketamine (50 mg/kg body weight), xylazine (5 mg/kg body weight), andacepromazine (0.75 mg/kg body weight). A second dose (20% of the firstdosage) was given IM 15 minutes before removal of vascular clip, andthird dose (60% of the first dosage) was given at tracheotomy.Intra-arterial catheter (22 G, 1 in. Angiocath) and intra-venouscatheter (24 G, 1 in. angiocath) were be placed in the ear centralartery and posterior marginal ear vein for blood samplings (arterialblood gases and CBC) and anti-IL-8 and fluid administration,respectively. The anaesthetized animals were transferred in a supineposition to an operating tray; the abdominal area was shaved andprepared for surgery. Via a midline laparotomy, the superior mesentericartery (SMA) was isolated and a microvascular arterial clip applied atthe aortic origin. Before the temporary closure of the abdomen using 9mm wound clip (Autoclip, Baxter), 15 ml of normal saline was givenintraperitoneally as fluid supplement. After 110 minutes of intestinalischemia, the abdominal incision was reopened and the arterial clip wasreleased to allow reperfusion. Before closure, 5 ml of normal saline wasgiven intraperitoneally for fluid replacement. The laparotomy incisionwas closed in two layers and the animals allowed to awaken.

After surgery, the animals were placed on a heating pad (38° C.) andcontinuously monitored for up to 6 hours post reperfusion and lactatedRinger's 8–12 ml/kg/hr IV was given as fluid supplement.

At 22–24 hr post-reperfusion, a tracheotomy was performed underanesthesia. Normal physiologic saline was diluted 1:3 with water andadjusted to pH 1.5 (adjusted by using 1N HCL); 3 ml/kg body weight wasthen instilled intra-tracheally. Rectal temperature was maintained at37+/−1 degree C. using a homeothermic heat therapy pad (K-Mod II,Baxter). Fluid supplements (LRS) at a rate of 5 ml/kg/hour IV weregiven. Blood gases were monitored every hour. The rabbits were returnedto the cage after 6 hr of continuous monitoring.

Just prior to aspiration, animals were treated with saline, the controlmonoclonal antibody (anti-HIV gp-120 IgG 9E3.1F10), the full lengthmurine anti-rabbit IL8 (6g4.2.5 murine IgG2a anti-rabbit IL8) or thepegylated 6G4V11N35A Fab′ (6G4V1N35A Fab′ modified with 40 kD branchedPEG-maleimide as described in Section T above, denoted as “40 kDbranched PEG-6G4V11N35A Fab′”). Data from saline or control antibodytreated animals was combined and presented as “Control”. Arterial bloodgases and A-a PO2 gradient measurements were taken daily, and IV fluidsupplementation was performed daily. A-a PO2 gradient was measured at 96hr of reperfusion. The A-a PO2 gradient was calculated as:A-a PO2=[FIO2(PB−PH2O)−(PaCO2/RQ)]−PaO2.

PaO2/FiO2 ratios were measured at 24 hr and 48 hr in room air and 100%oxygen.

After the final A-a PO2 gradient measurement, the animals wereanesthetized with Nembutal 100 mg/kg i.v. and the animals wereeuthanized by transecting the abdominal aorta in order to reduce redblood cell contamination of bronchoalveolar lavage fluid (BAL). Thelungs were removed en bloc. The entire lung was weighed and then lavagedwith an intratracheal tube (Hi-Lo tracheal tube, 3 mm) using 30 ml ofHBSS and lidocain. Total and differential leukocyte counts in the BALwere determined. Lesions/changes were verified by histologicalexamination of each lobe of the right lung of each animal.

The gross lung weight, total leukocyte and polymorphonuclear cell countsin BAL, and PaO2/FiO2 data obtained are depicted in FIGS. 67, 68 and 69,respectively. Treatment with 40 kD branched PEG-6G4V11N35A Fab′exhibited no effect on the biological parameters measured in the modelas compared to the “Control” group. However, the data do not contradictthe pharmacokinetic analysis or the in vitro activity analysis for the40 kD branched PEG-6G4V11N35A Fab′ presented in Sections (V) and (X)above. In addition, these data do not contradict the ability of the 40kD branched PEG-6G4V11N35A Fab′ to reach and act on disease effectortargets in circulation or other tissues.

Z. Additional In Vivo Efficacy Testing of Anti-IL-8 Antibody Reagents inRabbit Model of Ischemia/Reperfusion and Acid Aspiration-Induced AcuteRespiratory Distress Syndrome (ARDS)

Full length murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 and 20kD linear PEG-6G4V11N35E Fab′ were tested in a rabbit model ofischemia/reperfusion- and acid aspiration-induced acute respiratorydistress syndrome (ARDS).

Antibodies

A Fab′-SH antibody fragment of the affinity matured anti-IL-8 antibody6G4V11N35E was expressed using the Fab′ expression plasmid for6G4V11N35E (described in Section (T) above) in E. coli grown to highdensity in the fermentor as described by Carter et al., Bio/Technology,10: 163–167 (1992). Anti-IL-8 6G4V11N35E Fab′ variant was purified fromfermentation paste and modified with 20 kD linear methoxy-PEG-maleimideas described in Example T above. Pegylated material was formulated inphosphate buffered saline (PBS) at physiological pH. Full length 6G4.2.5antibody was obtained from hybridoma cell line 6G4.2.5 as described inSection (B) above and formulated in phosphate buffered saline (PBS) atphysiological pH.

Sterile Surgical Procedures and Post-Operative Care

Male New Zealand White rabbits weighing 2.2 to 2.5 kg (obtained fromWestern Oregon Rabbit Company) were anaesthetized by intramuscularinjection of ketamine (50 mg/kg body weight), xylazine (5 mg/kg bodyweight), and acepromazine (0.75 mg/kg body weight). Intra-arterialcatheter (22 G, 1 in. Angiocath) and intra-venous catheter (24 G, 1 in.angiocath) were be placed in the ear central artery and posteriormarginal ear vein for blood samplings (arterial blood gases and CBC) andanti-IL-8 (or fluid) administration, respectively. The anaesthetizedanimals were transferred in a supine position to an operating tray; theabdominal area was shaved and prepared for surgery. Via a midlinelaparotomy, the superior mesenteric artery (SMA) was isolated and amicrovascular arterial clip applied at the aortic origin. Before thetemporary closure of the abdomen using 9 mm wound clip (Autoclip,Baxter), 15 ml of normal saline (38° C.) was given intraperitoneally asfluid supplement. After 110 minutes of intestinal ischemia, theabdominal incision was reopened and the arterial clip was released toallow reperfusion. Before closure, 5 ml of normal saline (38° C.) wasgiven intraperitoneally for fluid replacement. The laparotomy incisionwas closed in two layers and the animals allowed to awaken.

After surgery, the animals were placed on a heating pad (38° C.) andcontinuously monitored for up to 6 hours post reperfusion and lactatedRinger's 8–12 ml/kg/hr IV was given as fluid supplement.

At 22–24 hr post-reperfusion, a tracheotomy was performed underanesthesia using ketamine, xylazine and acepromazine as described above.Normal physiologic saline was diluted 1:3 with water and adjusted to pH1.5 (adjusted by using 1N HCL), and 3 ml/kg body weight was theninstilled intra-tracheally through an uncuffed tracheal tube (2.0 mmI.D., Mallinckrodt Medical, Inc.). After instillation, the trachea wasclosed with 3-0 silk suture and the rabbits were allowed to recover.Rectal temperature was maintained at 37° C.+/−1° C. using a homeothermicheat therapy pad (K-Mod II, Baxter). Fluid supplements (LRS) at a rateof 5 ml/kg/hour IV were given. The rabbits were observed and blood gasesin room air and in 100% oxygen were measured daily.

Dose Administration

Treated animals received an intravenous injection of 7 mg/kg 20 kDlinear PEG-6G4V11N35E Fab′ (n=5 animals) at 10 minutes before and 6hours after acid instillation.

Oxygenation Measurement

Alveolar-arterial oxygen pressure gradient (A-a PO2 gradient) wascalculated as follows:A-a PO2=[FiO2(PB−PH2O)−(PaCO2/RQ)]−PaO2where FiO2 is fraction of inspired oxygen, PB is barometric pressure,PH2O is partial pressure of water vapor, PaCO2 is arterial carbondioxide pressure, RQ is respiratory quotient, and PaO2 is arterialoxygen pressure.

A-a PO2 gradient and PaO2/FiO2 ratios for each rabbit were measured atbaseline (pre-op), before acid instillation, every hour up to 6 hoursafter acid instillation, and every 24 hours thereafter.

Bronchoalveolar Lavage (BAL)

After blood gases measurement at 72 hours post reperfusion, the rabbitswere anesthetized with Nembutal 50 mg/kg i.v. and were euthanized byexsanguination. The abdominal aorta was transected to reduce red bloodcell contamination of bronchial alveolar lavage fluid (BALF). The lungand heart were removed en bloc. The right lung was lavaged with anintratracheal tube (Hi-Lo tracheal tube, 3.0 mm) using 20 ml of HBSS andlidocain. Total and differential leukocyte counts of BALF weredetermined.

Gross Lung Weight

The whole lung from each rabbit was weighed immediately after harvestand was expressed as g/kg of body weight.

Peripheral Blood Count

Blood samples (0.05 ml for CBC, 0.2 ml for blood gases) were collectedfrom the ear central artery catheter at baseline (pre-op), 2 hours, 4hours, 6 hours, and 22 hours post reperfusion (prior to acid or salineinstillation) and at 1 hour, 2 hours, 3 hours, 4 hours, 6 hours andevery 24 hours after acid instillation. Hematology parameters weredetermined by Automated Hematology Analyzer according to the standardhematological procedures.

Pharmacokinetics

Blood samples (0.5 ml) were collected from the ear central arterycatheter at baseline (pre-op), 4 hours, and 22 hours post reperfusionand at 1 hour, 4 hours, and every 24 hours after acid instillation.

Results and Discussion

In the rabbit model of ARDS, lung injury is manifested by hypoxemia (lowPaO2—the pressure of O2 in the arterial blood, as measured by a bloodgas machine), lung edema (evidenced by an elevated lung weight to bodyweight ratio) and pro-inflammatory in filtrates into the alveolar space(evidenced by high white blood cell (WBC) and neutrophil (PMN) numbers).Although 40 kD branched PEG-6G4V11N35A Fab′ did not protect rabbits fromlung injury at any of the doses tried (5 mg/kg and 20 mg/kg) (seeSection (Y) above), the 20 kD linear PEG-6G4V11N35E Fab′ had efficacyequal to, and, for some end-points, superior to that of the full lengthIgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 and preventedlung injury in the rabbits as shown in FIGS. 70A–70E. (The data pointsfor 40 kD branched PEG-6G4V11N35A Fab′ treated animals, full length6G4.2.5 treated animals, and saline treated animals appearing in FIGS.70A–70E are taken from the data displayed in FIGS. 67–69 and generatedin Example Y above.) In addition, these data indicate that largeeffective size anti-IL-8 Fab′-PEG conjugates can exhibit useful levelsof efficacy in acute lung injury and ARDS.

AA. In Vivo Efficacy Testing of Anti-IL-8 Antibody Reagents in RabbitEar Model of Tissue Ischemia and Reperfusion

Full length murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5, 20 kDlinear PEG-6G4V11N35E Fab′, 30 kD linear PEG-6G4V11N35E Fab′, and 40 kDbranched PEG-6G4V11N35E Fab′ were tested in a rabbit ear model of tissueischemia and reperfusion injury.

Antibodies

A Fab′-SH antibody fragment of the affinity matured anti-IL-8 antibody6G4V11N35E was expressed using the Fab′ expression plasmid for6G4V11N35E (described in Example T above) in E. coli grown to highdensity in the fermentor as described by Carter et al., Bio/Technology,10: 163–167 (1992). Anti-IL-8 6G4V11N35E Fab′ variant was purified fromfermentation paste and modified with 20 kD linear methoxy-PEG-maleimide,30 kD linear methoxy-PEG-maleimide, or 40 kD branchedmethoxy-PEG-maleimide as described in Example T above. Pegylatedmaterial was formulated in phosphate buffered saline (PBS) atphysiological pH.

Animals

1.0 to 1.5 kg New Zealand White rabbits were obtained from WesternOregon Rabbit Company.

Surgical Procedure and Animal Evaluation

The procedure was essentially described by Vedder et al., Proc. Natl.Acad. Sci. (USA), 87: 2643–2646 (1990). Briefly, general anesthesia wasachieved by intramuscular injections of Ketamine (50 mg/kg) plusXylazine (5 mg/kg) and Acepromazine (2 mg/kg). The right external earwas prepared for surgery and under sterile procedure the ear wastransected at its base, leaving intact only the central artery and vein.All nerves were transected to ensure that the ear was completelyanesthetic. A straight microaneurysm clip (1.5×10 mm) was placed acrossthe artery to produce complete ischemia. The ear was reattached with theclip exiting through the wound. The rabbits were then housed at 26° C.and 6 hours later the clip was removed to effect reperfusion. Untreatedrabbits (n=11 animals) received an intravenous injection of vehicle (10mM sodium acetate, 8% trehalose and 0.01% polysorbate-20 at pH 5.5)immediately prior to reperfusion. Treated animals received 5 mg/kg fulllength IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 (n=4animals), 20 kD linear PEG-6G4V11N35E Fab′ (n=3 animals), 30 kD linearPEG-6G4V11N35E Fab′ (n=3 animals), or 40 kD branched PEG-6G4V11N35E Fab′(n=3 animals) immediately prior to reperfusion.

The ear volume and necrosis were measured daily by procedures describedin Vedder et al., supra. Briefly, the ear was submerged in a beaker ofwater containing 1.2% Povidone iodine (Baxter) up to the intertragicincisure and the ear volume determined by the volume of fluid displaced.The ears were monitored in this manner for 7 days. The data arerepresented (in FIG. 71) as percent change in ear volume calculated asfollows:

${\%\mspace{14mu}{change}\mspace{14mu}{in}\mspace{14mu}{ear}\mspace{14mu}{volume}} = {\frac{\left( {{{Ear}\mspace{14mu}{{vol}.\mspace{14mu}{at}}\mspace{14mu}{day}\mspace{14mu} x} - {{Ear}\mspace{14mu}{{vol}.\mspace{14mu}{at}}\mspace{14mu}{day}\mspace{14mu} 0}} \right)}{{Ear}\mspace{14mu}{{vol}.\mspace{14mu}{at}}\mspace{14mu}{day}\mspace{14mu} 0} \times 100\%}$

Animals were sacrificed at day 1 and day 7 for histological evaluationof the ear and the same section of ear was taken from all animals.

To determine that the therapeutic agents did not adversely affect anyhematological parameter, aliquots of blood were withdrawn for completeblood counts and differentials immediately before reperfusion and at 24hour intervals. In a separate experiment, blood samples were taken at 1,5, 15, and 30 minutes and at 1 hour and 4 hours.

Results and Discussion

In the rabbit model of ear ischemia reperfusion injury, antibody wasadministered intravenously at a single dose (5 mg/kg) at the time ofreperfusion. In this model, ischemia reperfusion injury is characterizedby tissue damage, edema and sometimes necrosis; all attributable in partto neutrophil-mediated damage. Monitoring of ear volume over time is asurrogate end-point for evaluating edema in the ear tissue. Theresulting data (depicted in FIG. 71) showed that treatment with 20 kDlinear PEG-, 30 kD linear PEG- and 40 kD branched PEG-conjugated Fab'seffectively reduced ear swelling and edema at all time points ofobservation (days 1, 3 and 5). In fact, the efficacy of all threePEGylated Fab's was statistically indistinguishible from that of thefull length IgG murine anti-rabbit IL-8 monoclonal antibody 6G4.2.5 atall time points observed. These data support the efficacy of largeeffective size anti-IL-8 Fab′-PEG conjugates in ischemic reperfusioninjury and specifically support the ability of 40 kD branchedPEG-conjugated Fab′ molecules to reach and act on disease effectortargets in circulation and other tissues.

AB. Pharmacokinetic Studies of Two Constructs of Pegylated Anti-VEGFFab′ Fragments Following Intravenous and Intraperitoneal Administrationin Normal Mice

The objective of this study was to characterize and compare thepharmacokinetics of two pegylated species of the Y0317 affinity maturedvariant of humanized anti-VEGF F(ab)-12 (Y0317 is described in WO98/45331 published Oct. 15, 1998) (International Application No.PCT/US98/06604 filed Apr. 3, 1998) when administered intravenously andintraperitoneally in normal mice.

Methods

The Y0317 anti-VEGF Fab′ was obtained as described in Example 3 of WO98/45331. The Y0317 anti-VEGF Fab′ was pegylated with 20 kD linear PEGor 40 kD branched PEG using the thiol protection/deprotection maleimidecoupling method described in Example T above to form 20 kD linearPEG-Y0317 Fab′ and 40 kD branched PEG-Y0317 Fab′, respectively.Pegylated Fab's were formulated in phosphate buffered saline (PBS) atphysiological pH.

Male CD-1 mice (Charles River Laboratories, Hollister, Calif.), weighingbetween 18.9 and 28.8 grams, were injected with a single intravenous(IV) or intraperitoneal (IP) dose of either 20 kD linear or 40 kDbranched PEG-Y0317 Fab′ at approximately 3 mg protein/kg. Blood wascollected via cardiac puncture upon terminal sacrifice at the followingtime points (n=2 per time point): pre-dose, 5 and 30 minutes; 1, 2, 4,8, 24, 32 hours; Days 2, 3, 5, 7 10 and 14. Serum was harvested andanalyzed for PEGylated Y0317 Fab′ concentrations by ELISA. In addition,approximately 0.2 mL of serum was collected pre-dose from the Day 7 and14 mice (orbital bleed) as controls for potential antibody analysis.

For each PEGylated Y0317 Fab′, the pooled individual serum PEGylatedY0317 Fab′ concentration data following IV and IP administration wereanalyzed using a two-compartment and one-compartment pharmacokineticmodel, respectively. Concentration values that were below the lowerlimit of the assay were not used in the analysis.

Results

Table 11 below summarizes the compartmental pharmacokinetic parametersof the 20 kD linear and 40 kD branched PEG-Y0317 Fab′. FIGS. 1 and 2display the serum concentration versus time profiles following IV and IPadministration, respectively, for the 20 kD linear and 40 kD branchedPEG-Y0317 Fab′.

TABLE 11 Summary of PK parameters 20K 40K IV IP IV IP Dose (mg/kg) 3 33.4 3.4 Cmax (μg/mL) 58.1 11.5 74.6 49.8 Tmax 5 min 8 hr 30 min 4 hr CL(mL/day/kg) 179 236 49.4 37.0 t_(1/2) α (hr) 1.34 — 1.78 — t_(1/2) β(hr) 16.6 16.0^(a) 29.8 28.3^(a) AUC (day · μg/mL) 16.8 12.7 68.9 91.8Vss (mL/kg) 139 228 82.8 63.3 BA (%) — 75.6 — 133 ^(a)K10 half-life wasreported for IP.

After intravenous administration, the clearance of the 20 kD linearPEG-Y0317 Fab′ was 179 mL/day/kg and decreased to 49.4 mL/day/kg withthe 40 kD branched PEG-Y0317 Fab′. Vss, also decreasing with a largerPEG size, was 139 and 82.8 for the 20 kD and 40 kD PEGylated species,respectively. In accord with the decrease in clearance and volume ofdistribution, an increased terminal half-life was observed for thelarger PEG size (terminal t_(1/2) of approximately 17 and 30 hours forthe 20 kD and 40 kD PEGylated species, respectively). After IPadministration, the bioavailability was approximately 76 and 133% forthe 20 kD and 40 kD PEGylated species, respectively. Results fromprevious pharmacokinetic studies with intravenous administration of ananti-CD18 Fab in a normal mouse model (Zapata et al., “Site SpecificCoupling of Monomethoxypoly(ethylene) glycol to a single-sulfhydrylhumanized Fab′”, poster presented at the American Society forBiochemistry and Molecular Biology FASEB Meeting held in San Francisco,Calif. on May 21–25, 1995; Abstract No. 1288 published in Zapata et al.,FASEB J., 9(6): A1479 (1995)) indicated that the clearance of anon-PEGylated Fab′ was approximately 4500 mL/day/kg and the terminalhalf-life was approximately 30 minutes. Taken together, these dataindicate that 20 kD (linear) and 40 kD (branched) PEGylation of theY0317 Fab′ resulted in an approximately 25-fold to 90-fold decrease inclearance and a 29-fold and 52-fold increase in terminal t_(1/2),respectively.

AC. In Vivo Efficacy Testing of Anti-VEGF Antibody Reagents in MouseModel of Tumor Growth

40 kD branched PEG-Y0317 anti-human VEGF Fab′ was tested in a mousetumor growth model.

Methods

40 kD branched PEG-Y0317 anti-human VEGF Fab′ was obtained as describedin Example AB above. Y0317 anti-human VEGF MAb (full length IgG1) wasobtained by fusing the Y0317 variable light (V_(L)) and variable heavy(V_(H)) domain sequences to constant light (CL) and constant heavy (CH)domain sequences, respectively, in separate pRK expression vectors asdescribed in Eaton et al., Biochemistry, 25: 8343–8347 (1986),co-transfecting the expression constructs into 293 cells or CHO cells,and harvesting antibody from transfected cell culture supernatantessentially as described in Example 1 of WO 98/45331 (published Oct. 15,1998) (International Application No. PCT/US98/06604 filed Apr. 3, 1998).Control 40 kD branched PEG-6G4V11N35E anti-rabbit IL-8 Fab′ was obtainedas described in Example AA above.

Human A673 rhabdomyosarcoma cells (ATCC; CRL1598) were cultured aspreviously described in DMEM/F12 supplemented with 10% fetal bovineserum, 2 mM glutamine and antibiotics (Kim et al. Nature 362:841–844(1993) and Borgström et al. Cancer Res. 56:4032–4039 (1996)). FemaleBeige nude mice, 6–10 weeks old (Harlan Sprague Dawley), were injectedsubcutaneously in the dorsal area with 2.5×10⁶ A673 tumor cells in avolume of 100 μl of matrigel. Animals were then treated with 40 kDbranched PEG-Y0317 Fab′, Y0317 MAb, control 40 kD branchedPEG-6G4V11N35E anti-rabbit IL-8 Fab′, or phosphate buffered saline (PBS)at physiological pH.

In the low dose 40 kD branched PEG-Y0317 Fab′ treatment group, 40 kDbranched PEG-Y0317 Fab′ was administered at a loading dose of 2 mg/kg onday 1, followed by maintenance doses of 0.9 mg/kg daily for theremainder of the study.

In the high dose 40 kD branched PEG-Y0317 Fab′ treatment group, 40 kDbranched PEG-Y0317 Fab′ was administered at a loading dose of 6 mg/kg onday 1, followed by maintenance doses of 2.7 mg/kg daily for theremainder of the study.

In the Y0317 MAb treatment group, Y0317 MAb was administered at aloading dose of 8 mg/kg on day 1, followed by maintenance doses of 0.8mg/kg every third day for the remainder of the study.

In the control Fab′ treatment group, 40 kD branched PEG-6G4V11N35Eanti-rabbit IL-8 Fab′ was administered at a loading dose of 6 mg/kg onday 1, followed by maintenance doses of 2.7 mg/kg daily for theremainder of the study.

In the PBS control group, 0.1 ml/day of PBS was administered for theduration of the study. All doses were administered intraperitoneally ina volume of 100 μl, starting 24 hr after tumor cell inoculation.

Each group initially consisted of 10 mice. Tumor size(length×width×height) was determined at weekly intervals. 17 days aftertumor cell inoculation, animals were euthanized and the tumors wereremoved and weighed. Statistical analysis was performed by ANOVA.

Results

As shown in FIG. 74, at both doses tested (2 and 6 mg/kg), the 40 kDbranched PEG-Y0317 Fab′ markedly suppressed tumor growth as assessed bytumor weight measurements three weeks after tumor cell inoculation. Thedecreases were 91% and 90%, respectively, in animals treated with thelow and high doses of 40 kD branched PEG-Y0317 Fab′ versus 95% inanimals treated with Y0317 MAb.

The following biological materials have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC):

ATCC Material Accession No. Deposit Date hybridoma cell line 5.12.14 HB11553 Feb. 15, 1993 hybridoma cell line 6G4.2.5 HB 11722 Sep. 28, 1994pantiIL-8.2, E. coli strain 294 mm  97056 Feb. 10, 1995 p6G425chim2, E.coli strain 294 mm  97055 Feb. 10, 1995 p6G4V11N35A.F(ab′)₂  97890 Feb.20, 1997 E. coli strain  98332 Feb. 20, 1997 49D6(p6G4V11N35A.F(ab′)₂)p6G425V11N35A.choSD 209552 Dec. 16, 1997 clone#1933 aIL8.92 NB 28605/12CRL-12444 Dec. 11, 1997 clone#1934 aIL8.42 NB 28605/14 CRL-12445 Dec.11, 1997

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable deposit for 30 years fromthe date of deposit. These cell lines will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the cell lines to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the cell lines to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the depositedcell lines should be lost or destroyed when cultivated under suitableconditions, they will be promptly replaced on notification with aspecimen of the same cell line. Availability of the deposited cell linesis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

1. A conjugate consisting essentially of an antibody fragment covalently modified by one or two nonproteinaceous polymer molecules at a free sulfhydryl group of a cysteine residue within the hinge region of the antibody fragment, wherein a disulfide bridge within the hinge region is avoided by substituting another amino acid for the corresponding cysteine reside within the hinge region in the opposite chain of said antibody fragment, and (a), wherein the apparent molecular weight of the conjugate, as determined by size exclusion chromatography is about 500 kD, (b) the average actual molecular weight of each nonproteinaceous polymer molecule is at least 20 kD, (c) the conjugate has an apparent size that is about 8-fold greater than the apparent size of the parental antibody fragment, (d) the conjugate binds the same antigen as the parental molecule that is not covalently modified by one or two nonproteinaceous polymer molecules, and wherein the antibody fragment comprises an antigen binding site that binds to a polypeptide selected from the group consisting of human vascular endothelial growth factor (VEGF), human CD18, human CD11a, human IgE, human Apo-2 receptor, human tumor necrosis factor-α (TNF-α), human tissue factor (TF), human α₄β₇ integrin, human GPIIb-IIIa integrin, human epidermal growth factor receptor (EGFR), human CD3, and human interleukin-2 receptor α-chain (TAC).
 2. The conjugate of claim 1, wherein the apparent size of the conjugate is about 800 kD.
 3. The conjugate of claim 1, wherein the apparent size of the conjugate is about 1,400 kD.
 4. The conjugate of claim 1, wherein the apparent size of the conjugate is about 1,800 kD.
 5. The conjugate of claim 1, wherein the apparent size of the conjugate is about 15 fold greater than the apparent size of the parental antibody.
 6. The conjugate of claim 5, wherein the apparent size of the conjugate is about 25 fold greater than the apparent size of the parental antibody.
 7. The conjugate of claim 1 wherein the antibody fragment is selected from the group consisting of Fab′, Fab′-SH, and F(ab′)₂.
 8. The conjugate of claim 7 wherein the antibody fragment is F(ab′)2.
 9. The conjugate of claim 1 wherein the antibody fragment is attached to no more than 1 nonproteinaceous polymer molecule.
 10. The conjugate of claim 1 wherein at least one nonproteinaceous polymer is a polyethylene glycol (PEG).
 11. The conjugate of claim 10 wherein the PEG has an average molecular weight of about 20 kD.
 12. The conjugate of claim 11 wherein the PEG has an average molecular weight of about 40 kD.
 13. The conjugate of claim 10 wherein the PEG has an average molecular weight of about 30 kD.
 14. The conjugate of claim 10 wherein the PEG is a single chain molecule.
 15. The conjugate of claim 10 wherein the PEG is a branched chain molecule.
 16. The conjugate of claim 1 wherein the antibody fragment comprising the antigen binding site is humanized.
 17. The conjugate of claim 7 wherein the antibody fragment is Fab′.
 18. The conjugate of claim 7 wherein the antibody fragment is Fab′-SH.
 19. The conjugate of claim 1 wherein the antibody fragment is attached to two nonproteinaceous polymer molecules.
 20. The conjugate of claim 19, wherein the two nonproteinaceous polymer molecules are PEG molecules. 